Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology
- Matthias Gehringer*Matthias Gehringer*Phone: +49 7071 29-72466. E-mail: [email protected]Department of Pharmaceutical/Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, GermanyMore by Matthias Gehringer and
- Stefan A. LauferStefan A. LauferDepartment of Pharmaceutical/Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, GermanyMore by Stefan A. Laufer
Copyright © 2018 American Chemical Society
Abstract
Targeted covalent inhibitors (TCIs) are designed to bind poorly conserved amino acids by means of reactive groups, the so-called warheads. Currently, targeting noncatalytic cysteine residues with acrylamides and other α,β-unsaturated carbonyl compounds is the predominant strategy in TCI development. The recent ascent of covalent drugs has stimulated considerable efforts to characterize alternative warheads for the covalent-reversible and irreversible engagement of noncatalytic cysteine residues as well as other amino acids. This Perspective article provides an overview of warheads—beyond α,β-unsaturated amides—recently used in the design of targeted covalent ligands. Promising reactive groups that have not yet demonstrated their utility in TCI development are also highlighted. Special emphasis is placed on the discussion of reactivity and of case studies illustrating applications in medicinal chemistry and chemical biology.
1. Introduction
ARTICLE SECTIONS
Covalent targeting has played a subordinate role in drug discovery for a long time. Although covalent modification of proteins(1,2) and nucleobases(3) is a key element in the regulation of biological systems, the systematic development of reactive drugs has been considered highly adventurous because of potential toxicity arising from promiscuous labeling, haptenization, and idiosyncratic drug reactions.(4,5) Nevertheless, over 40 covalent modifier drugs are currently approved by the FDA.(6) However, the mode of action of most of these compounds has been discovered serendipitously rather than resulting from rational design. Among the notable exceptions are inhibitors of amide-/ester-bond cleaving hydrolases, where reversible and irreversible covalent bond formation has long been used as a design strategy to address catalytic nucleophiles such as activated serine or cysteine residues.(7,8) During the past decade, covalent targeting has experienced a resurgence. So-called “targeted covalent inhibitors” (TCIs) addressing poorly conserved amino acids now provide the basis for a multitude of industrial drug discovery programs, especially in oncology.(5) According to the definition from a seminal review by Juswinder Singh and colleagues, a TCI is “an inhibitor bearing a bond-forming functional group of low reactivity that, following binding to the target protein, is positioned to react rapidly with a specific noncatalytic residue at the target site.”(5) Naturally, this concept can further be extended to other types of targeted covalent ligands beyond (enzyme) inhibitors. The renewed interest in covalent targeting is based on the perception that properly designed TCIs could offer multiple advantages, some of which would even result in a more favorable benefit–risk balance. Irreversible covalent inhibitors profit from nonequilibrium kinetics, potentially enabling full target occupancy, even if reversible binding affinity is moderate. In contrast to reversible binders, irreversibly bound compounds do not compete with natural ligands or substrates. Their durable target engagement can further decouple pharmacodynamics from pharmacokinetics provided that the protein of interest has a sufficiently slow rate of resynthesis.(6) The driving force and spatial requirements of covalent bond formation can be exploited to reduce the size of ligands without sacrificing potency and selectivity, thereby increasing “drug-likeness.” Considering that a substantial fraction of the drug candidates fail due to ADME issues,(9) the above features promise significant benefits. In cases where permanent protein modification is undesired, covalent-reversible chemistry can be used to harness potential advantages in terms of potency and selectivity while decreasing the risk of haptenization, inhibitor depletion by glutathione (GSH), and irreversible off-target modification.(10) The residence times of covalent-reversible inhibitors strongly depend on reversible interactions stabilizing the covalent complex. Thus, they can be tuned to fit the target’s turnover rate.(11) Ideally, the unmodified ligand would be released to re-engage with the target after protein degradation or after nonspecific covalent binding to off-targets incapable of stabilizing the covalent complex.
TCIs are typically generated by structure-based design from optimized reversible ligands, which are modified by attachment of an electrophilic covalent reactive group(12) (CRG; often termed “warhead”) to address a proximal amino acid, most frequently cysteine. Alternative strategies that have recently been employed to identify covalent binders include fragment-based(13−16) or tethering approaches(17) and DNA-encoded libraries featuring electrophilic ligands.(18,19) In TCI design, binding kinetics require special consideration. Because a detailed perspective on the implications of covalent binding kinetics has recently been provided,(20) only a brief overview shall be given at this place. TCI binding involves a two-step process in which an initial reversible binding event takes place, followed by the covalent bond-forming reaction. The first step is described by the binding constant KI, which accounts for the ligand concentration required to achieve a half-maximal rate of covalent modification. The second step is characterized by kinact, the maximal potential rate of covalent modification/inactivation. The overall efficiency of covalent target modification is best described as the second-order rate constant kinact/KI.(21) As a result, covalent inhibition is time-dependent and prolonged exposure leads to an increase in target occupancy. Consequently, KD, IC50, or EC50 values are not the appropriate measures for comparing covalent ligands because they vary over time and do not reflect the relative contribution of KI and kinact to the observed overall effect. Notably, time-dependent inhibition can also result from other factors like compound aggregation or instability in buffer and time-dependence cannot be considered as an ultimate proof of covalent binding despite the presence of a reactive group. On the other hand, very slow off-rate ligands may also be misinterpreted as covalent binders in washout and jump dilution experiments.(20) Beside the investigation of binding kinetics, cross-validation of the presumed covalent interaction, preferably by both mass spectrometry and X-ray crystallography, should thus be performed. Ideally, the latter data would be supported by studies with the mutated target protein devoid of the reactive amino acid and with congeneric ligands lacking the CRG. The two-step binding mechanism of TCIs differs from the one of reagents used for protein labeling or bioconjugation. The latter are used for chemo- but not site-selective covalent modification and preferably address the most reactive and accessible side chains. In this case, the reaction can be described by a one-step mechanism(20) without the requirement of specific reversible binding. Here, comparably reactive moieties relying solely on their intrinsic chemical reactivity toward the desired amino acids are used. Warheads to be utilized in TCIs require a more balanced reactivity profile. They should be reactive enough to form a covalent bond with the target residue in a proximity driven or templated manner, but only after the proper alignment of both reaction partners by the reversible binding event. Inherent reactivity should be reduced to a necessary minimum to prevent nonspecific off-target labeling or reaction with GSH. Because the reactivity of amino acid side chains varies as a function of their chemical environment, the reactivity of ideal CRGs should be readily adjustable to match the specific requirements of the target. Warheads intended for in vivo use, either in drugs or in chemical probes, should be chemically and (usually) metabolically stable and nontoxic. If chemical probes are intended for the investigation of specific targets or pathways in cells, some degree of chemical instability or toxicity might be tolerable. The latter class of ligands should be as selective as possible, which is not necessarily true for drugs. In contrast, reactive probes for activity-based protein profiling approaches (ABPP)(22) require a balanced amount of promiscuity to enable the profiling of a defined set of targets.
As a result of the time-dependent nature of covalent target engagement, even optimized compounds, which have demonstrated good selectivity in screening panels, can cause significant off-target labeling after extended exposure.(23,24) On the other hand, relatively small, reactive, and presumably unselective fragments can possess an unexpected degree of specificity when profiled against isolated proteins(15) or cellular proteomes.(13) The latter findings suggest that an increased reactivity might be exploited to achieve kinetic selectivity if the reactive moiety was completely neutralized by the target or metabolically deactivated after having engaged the target (vide infra). Certain highly reactive compounds such as α-cyanoacrylamide-derived Michael acceptors can further be useful for covalent-reversible targeting approaches.(25) Finally, the ideal balance of reversible binding affinity and specificity, inherent reactivity, and metabolic stability depends on the envisaged field of application and needs to be evaluated on a case by case basis.
There has been a very high interest in novel TCIs within the last years, the tremendous amount of published work having been compiled in a plethora of recent reviews.(5,6,26−39) Most of these reports focus primarily on noncatalytic cysteine residues addressed by α,β-unsaturated carbonyl compounds. Although cysteine-targeted Michael acceptors, most notably α,β-unsaturated amides, are clearly the dominant CRGs in the realm of current TCIs, they are not intended to be a key subject in this essay. An overview of α,β-unsaturated carbonyl compounds has already been provided in a recent Perspective article by Kay Brummond, Daniel Harki, and co-workers, extensively discussing the chemistry and reactivity of Michael acceptors as well as their application in drug discovery and beyond.(40) Instead, this Perspective focuses on less common warheads, recent innovations, and emerging concepts, highlighting case studies to illustrate their potential in medicinal chemistry and chemical biology. Reagents for protein modification, which have also been reviewed recently,(41−47) are not included except for certain functional groups where utility of the underlying reactivity in TCI design is anticipated. Likewise, warheads that have exclusively been used to target highly reactive active site nucleophiles, e.g., catalytic serine, threonine, or cysteine residues in proteolytic enzymes, are not a major focus of this Perspective and may be found elsewhere.(7,8,48) As an exception, few moieties engaging catalytic nucleophiles are discussed to highlight the underlying reactivity or design principles. It should further be pointed out that many warhead classes included in this article have previously found application in targeting the active sites of proteases and other hydrolase enzymes.
Despite the enormous amount of work that has been published on acrylamide-derived TCIs, systematic studies evaluating warheads beyond α,β-unsaturated amides are comparably sparse and scattered. Although we aimed to include all relevant and recent information, this work should rather be considered a (personally biased) perspective on interesting chemistry and current developments and not a comprehensive review of the field. This article covers literature published before July 2018, therefore interesting work which appeared during the revision of the manuscript (e.g., cysteine-targeted cyanamides as Janus kinase 3 inhibitors,(49) histidine-targeted linear alkyl bromides as 17β-hydroxysteroid dehydrogenase inhibitors,(50) and methionine-targeted epoxides as bromodomain inhibitors(51)), is not discussed. The article is organized by reactive amino acid side chains. Thus, warheads that have been used to target different residues may occur in several sections. (Pseudo)irreversible CRGs are discussed first, while covalent-reversible warheads may be found at the end of each section. A brief personal perspective on benefits and challenges of the respective CRG is given in each paragraph, and broader discussion may be found in the final Perspective section. Being a major subject of this article, inherent warhead reactivity and reaction rates are only compared directly if they were tested in the same assay. Otherwise, only a qualitative assessment is provided. Notably, there is still a heavy reliance on IC50/EC50 data in the field, although kinact/KI is the recommended measure for covalent binders. We have included kinetic data whenever it was available, but IC50 values are the basis for discussion if no such data has been reported.
2. Targeting the Cysteine Side Chain
ARTICLE SECTIONS
Sulfur is the only third-row element encoded in proteinogenic amino acids and has a distinct role in biological systems. The thioether group in methionine and the neutral cysteine thiol group are only moderately nucleophilic. However, nucleophilicity is increased by several orders of magnitude for the thiolate form of the cysteine side chain, making it the strongest nucleophile among the 20 canonical amino acids.(52,53) Being highly polarizable, thiols and thiolates are soft bases according to Pearson’s hard and soft acids and bases (HSAB) concept.(54) The increased nucleophilicity and polarizability of thiolates compared to alkoxides is owed to the size and the high energy of the 3sp3 lone pairs. The thiol group can easily be deprotonated (pKa ≈ 8.6 for cysteine), and the pKa can shift several orders of magnitude within proteins.(55) While pKa values between 2.5 and 11.1 have been measured for cysteines’ thiols in proteins,(56) noncatalytic cysteine residues typically feature pKa values in the range between 7.4 and 9.1. Catalytic cysteines, in contrast, are pKa-perturbed to favor the highly nucleophilic thiolate anion.(55) Therefore, it is hardly surprising that cysteine has various roles in catalysis, e.g., in the active sites of cysteine proteases, ubiquitin ligases, or tyrosine phosphatases, but also in metal binding, structural stabilization (via the formation of disulfide bridges), and posttranslational/redox regulation.(57,58) Notwithstanding these key functions, cysteine is a relatively rare amino acid with a prevalence of only 1.9%.(59) Hence, noncatalytic cysteine residues played a key role in recent TCI design efforts. For example, all the six currently approved targeted covalent kinase inhibitors address an equivalently positioned, poorly conserved cysteine in the solvent-exposed front region of ErbB/HER family receptor tyrosine kinases or TEC family kinases like Bruton’s tyrosine kinase (BTK) via acrylic or butynoic amides.(35,60,61) An analogous cysteine has been used to generate highly selective Janus kinase (JAK) 3 inhibitors with a promising potential in the treatment of inflammatory disorders.(62) Besides TCIs, endogenous ligands such as cyclopentenone prostaglandins,(63) but also a multitude of other natural α,β-unsaturated carbonyl compounds,(40) covalently engage noncatalytic cysteines. As suggested by the differing pKa values, cysteine reactivity can vary over a wide range, and not all exposed cysteine residues may be readily amenable to covalent modification.(64) Targeting less reactive cysteine residues is not always straightforward, and success may crucially depend on precise positioning and sufficient reactivity of the CRG but also on assistance by the CRG or neighboring groups in deprotonating the thiol. Moreover, nonessential cysteines are prone to mutation (see for example the EGFR C797S resistance mutant)(65) which can be considered an Achilles’ heel of cysteine-targeted covalent drugs, especially in oncology. On the other hand, acquired cysteine residues (e.g., in the oncogenic K-Ras G12C or the p53 Y220C mutants) may open up new avenues for addressing challenging targets.
2.1. Cysteine Addition to Metabolically Labile Monomethyl Fumarates
Although targeted covalent inhibitors derived from α,β-unsaturated amides are not a major topic here, some recent “noncanonical” approaches merit further discussion. As mentioned, TCIs often feature excellent selectivity for their targets upon short-term treatment but extended exposure has been shown to erode selectivity.(23,66) Slower covalent modification of off-targets can be missed by classical screening techniques typically investigating short-term effects (minutes to few hours). A possible solution to this issue is provided by covalent-reversible binders, such as the above-mentioned α-cyanoacrylamides. Another interesting approach to minimize time-dependent off-target modification has recently been developed in Benjamin Cravatt’s laboratories. While investigating the cysteine labeling profile of the immunomodulatory drug dimethyl fumarate (DMF) in T cell proteomes, they found the active metabolite monomethyl fumarate (MMF) to be devoid of substantial cysteine reactivity,(67) a finding that can be attributed to the decreased intrinsic reactivity of monomethyl fumarate but that might also reflect the presumably lower cell permeability of MMF. On the basis of this observation, the hypothesis arose that replacement of a conventional α,β-unsaturated amide in a cysteine-targeted inhibitor by a methyl fumarate residue could furnish compounds that rapidly react with their targets while the slower inactivation by esterases prevents off-target modification (Figure 1).
Figure 1
Figure 1. Kinetic selectivity of fumaric acid esters. Selectivity is povided by rapid bond formation with the target. Slightly slower ester cleavage deactivates the warhead, preventing even slower labeling of undesired proteins.
To test this hypothesis, the approved covalent Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib(68) (1, Figure 2), which is known to react with off-targets in a time-dependent manner,(23) was chosen for a first proof of concept study. Ibrutinib features a piperidin-3-yl-linked acrylamide warhead that labels BTK at Cys481.(69,70) The latter was replaced by several amide-linked fumarates (3–7). Some of the studied compounds (2 and 5–7) were further equipped with an alkyne to provide a chemical handle enabling the attachment of tags for enrichment and target identification. Both ibrutinib-derived probe 2 and its methyl fumarate analogue 5 labeled BTK in Ramos cell lysates. As expected, fumarate 5 reacted faster with cysteine and possessed the higher reactivity toward the cellular proteome. When incubated with HEK293T cells stably expressing human carboxylesterase (hCES) 1, ibrutinib remained unaffected while the methylfumarate-derived inhibitor 3 was rapidly converted to the unreactive acid 4. This effect was not observed when using HEK293T cells stably expressing methionine aminopeptidase (MetAP) 2 as a control. A significant reduction in time-dependent proteome labeling accompanied by a modest decrease in BTK activity (ca. 10-fold) was observed for probe 5 when incubated over 24 h with a 6:1 coculture of Ramos and HEK293T cell lines, which was supposed to mimic the hCES1 activity in tumor xenografts. In contrast, the labeling profile of the ibrutinib-derived probe 2 remained unaffected under these conditions. Competition experiments employing gel-based or MS proteomics(71) confirmed that BTK engagement by inhibitor 3 was only marginally affected by hCES1, supporting the notion that kinase modification occurs at a higher rate than enzymatic ester hydrolysis. Only BTK and the kinase TEC, sharing an equivalently positioned cysteine,(62) reacted with 3 in a hCES1-insensitive manner while 1 retained its proteomic profile in the presence of the esterase.
Figure 2
Figure 2. Ibrutinib-derived fumarate esters and analogous probes equipped with a click handle.
Subsequent profiling of probe 5 and its isopropyl ester analogue 6 in rodents (20 mg/kg ip) demonstrated BTK engagement in vivo, although both compounds were poorly stable in mouse plasma (t1/2 = < 2 min). Pretreatment with the covalent CES inhibitor JZL184,(72) however, increased the plasma stability to 25.5 and 352 min for 5 and 6. In contrast, probe 2 and the free acid 7 did not require CES blockage to obtain reasonable plasma stability (t1/2 = 168 and 129 min). Both 5 and 6 demonstrated significantly reduced off-target labeling in different tissues while substantial reactivity toward BTK was maintained. Although these results are qualitative in nature, they underline the increased BTK selectivity of the fumaric acid esters in vivo.
Although the metabolic inactivation of the reactive species to promote kinetic selectivity is a promising concept, the generalizability of this approach remains to be shown. Alternative esters might be required to fine-tune stability against esterases, and those will need to fit the respective binding pockets. As suggested by the authors of the above study, alternative warheads with limited metabolic stability such as acrylates or thioacrylates may have the potential to address these issues.(24) However, optimization of such compounds for clinical use, especially when considering oral administration will further complicate the situation due to the prolonged and more complex kinetics of uptake and distribution as well as first-pass metabolism. Moreover, proteins with a high turnover constitute a challenge to this conceptual framework as they require sustained exposure for pharmacological efficacy. Nevertheless, this study demonstrates that kinetic selectivity is achievable with metabolically labile warheads and it will be interesting to see whether related concepts will expand our current toolbox for TCI design.
2.2. Cysteine Addition to Allenamides
Recently, allenamides have been suggested as bioisosteres of α,β-unsaturated amides for protein labeling purposes and inhibitor design. Although the reactivity of allenamides (general structure 8, Figure 3) toward thiols has been known for a long time,(73,74) Teck-Peng Loh and colleagues were the first to investigate these reagents for cysteine labeling applications.(75) They found a high intrinsic reactivity toward the thiol(ate) of isolated cysteine as well as for terminal and internal cysteine residues in peptides and proteins at pH 8. However, neither were reaction rates determined nor was a qualitative comparison of reactivity between aniline-derived allenamides and alkylamine-derived analogues provided. No labeling of other nucleophilic amino acids was observed. Upon reaction with cysteine at the sp-hybridized carbon center, only the thermodynamically disfavored nonconjugated product 10a was formed, which is probably due to prevalence of the mesomeric structure 9a favoring protonation in the α-position. Notably, a similar regioselectivity was recently observed for selenocysteine conjugation.(76) Compared to their keto and ester analogues, terminal allenamides have a much lower tendency to undergo [3 + 2] cycloaddition reactions and cysteine labeling was shown to be irreversible even in the presence of a 100-fold excess of glutathione or dithiothreitol (DTT).
Figure 3
Figure 3. Mechanism of cysteine addition to allenamides. The prevalence of the mesomeric structure 9a rationalizes the formation of the nonconjugated product. An alternative mechanism involving attack of the neutral thiol to form a zwitterionic species followed by proton transfer was proposed by Loh and co-workers.
While the Loh group only tested this CRG for protein labeling, a subsequent study by Yujun Zhao and colleagues exploited the allenamide moiety as a bioisosteric replacement of the acrylamide functionality in the approved EGFR inhibitor osimertinib (11, Figure 4).(77) The allenamide warhead was well tolerated in terms of potency, and analogue 12 was slightly more active in blocking the EGFR T790M/L858R mutant compared to the parent compound (IC50 = 1.4 nM vs 3.9 nM for EGFRT790M/L858R). However, the compound partially eroded osimertinib’s selectivity over the wild-type enzyme (IC50 = 28 nM vs 142 nM for EGFRwt). Further optimization furnished low nanomolar inhibitors with increased (up to 43-fold for compound 13) or decreased (only 6-fold for compound 14) selectivity over the wild-type enzyme. Selectivity in the enzyme assay, however, did not translate into cellular selectivity for NCI-H1975 (EGFRL858R/T790M) versus A549 (EGFRwt) cells. Target engagement in NCI-H1975 cells was suggested by Western blot, but neither was covalent binding unambiguously demonstrated nor were off-targets assessed. Allenamide 12 showed poor oral exposure in mice, and both 12 and 14 possessed an over 10-fold lower stability in fetal bovine serum compared to osimertinib. The decreased stability is likely to result from the higher intrinsic reactivity of the allenamide warhead compared to osimertinib’s acrylamide. Pseudo-first-order rates for the reaction of compound 14 with glutathione (k′ = 0.303 min–1) were shown to be 7–28-fold higher than for the approved acrylamide-derived kinase inhibitors afatinib (k′ = 0.044 min–1), osimertinib (k′ = 0.011 min–1), and ibrutinib (k′ = 0.011 min–1). Although terminal allenamides offer certain advantages such as good accessibility, irreversibility, compact size, and a well-defined geometry, their utility in medicinal chemistry remains thus limited by the high intrinsic reactivity. In analogy to α,β-unsaturated amides,(12) a decrease in reactivity might be achieved by employing alkylamine-derived amides. In certain cases, for example when poorly nucleophilic cysteines(78) need to be addressed, the higher reactivity may provide a benefit, assuming that the reversibly binding part of the inhibitor confers sufficient kinetic selectivity to minimize off-target labeling.
Figure 4
Figure 4. Osimertinib-derived allenamides as EGFR inhibitors.
2.3. Cysteine Addition to Linear 3-Aryl and Alkyl Propiolonitriles
Alain Wagner and co-workers have recently established 3-aryl propiolonitriles (general structure 15, Figure 5A) as reagents for selective cysteine modification in peptide mixtures.(79) The obtained vinyl thioether adducts were stable against thiols over a wide pH range (from 0–14), reducing agents (tris(2-carboxyethyl)phosphine (TCEP) and DTT), and a reasonable stability was also demonstrated in plasma and living cells. In contrast, structurally related aryl alkynones undergo thiol exchange via addition of a second thiol and subsequent elimination of one thiolate (Figure 5B).(80) Reactivity of unsubstituted 15 is presumably too high for medicinal chemistry applications. However, reactivity could be decreased by substituents with a +M effect in the para-position (and vice versa, with substituents featuring a −M effect) and dropped substantially when ortho-substituents were introduced. Moreover, the analogous 3-cyclohexylpropiolonitrile (16) reacted about 10 times slower compared to unsubstituted 15, however, the obtained reaction product also proved to be slightly less stable. Although no applications in a medicinal chemistry or chemical biology context have been discussed so far, this linear electrophile could become very useful when precise and rigid spatial placement of the warhead is required (provided that the binding cavity can host this elongate functionality). Still, it remains to be seen if this CRG can be attenuated to an appropriate intrinsic reactivity to become useful for chemical probe development.
Figure 5
Figure 5. Propiolonitriles as potential TCI warheads: (A) 3-Aryl and 3-alkyl propiolonitriles. (B) Mechanism of cysteine addition and thiol exchange.
2.4. Cysteine Addition to Alkenyl or Alkynyl-Substituted Heteroarenes
Electron-deficient heteroaryl groups with (substituted) vinyl or ethynyl residues constitute a structural motif frequently found in patent applications claiming covalent inhibitors. These moieties are supposed to react with thiols via a conjugate addition, in which the intermediate negative charge is stabilized by the heteroarene. This reaction type has been known for a long time. For example, 4-vinylpyridins have been used to modify cysteines since the 1960s(81,82) and chemical reactions exploiting alkenylated or alkynylated aromatic heterocycles for nucleophilic additions are not uncommon (e.g., refs (83−88)). Vinylpyridines have also been applied in cysteine-specific protein labeling.(89,90) and their reactivity can be readily modulated.(91) In this light, it is surprising that only a few and relatively limited systematic studies evaluating this chemistry for cysteine-targeted inhibitors have been published so far.
An early isolated series of publications on the application of such CRGs in medicinal chemistry was reported by David Uehling and co-workers from GSK in 2008/2009.(92) They serendipitously found a noncatalytic cysteine of ErbB kinases to react with ethynylthienopyrimidine-based inhibitors. Although the ethynyl moieties were attached to the thiophene ring, which is usually electron-rich, covalent bond formation with Cys803 in ErbB4 and Cys797 in EGFR was observed. In a subsequent SAR study, the influence of a terminal substituent at the ethynyl moiety was evaluated for both a 6-ethynylthieno[3,2-d]pyrimidine (17–20, Figure 6A) and a 6-ethynylthieno[2,3-d]pyrimidine series (exemplified by compound 21). Consistent with a mechanism in which the thiol(ate) attacks the triple bond generating a transient negative charge, the 6-ethynylthieno[3,2-d]pyrimidine-derived compounds were more reactive due to better resonance stabilization of the latter. However, labeling was generally slow. While the acrylamide-derived control compound canertinib (CI-1033)(93) completely labeled EGFR within less than 3 h, only 67% of covalent modification was observed for the terminal alkyne 17 after 20 h as determined by MS. Interestingly, the attachment of a 2-(R)-pyrrolidine residue (18) significantly accelerated covalent inactivation, leading to 83% labeling after 3 h and full labeling after 20 h, respectively. This acceleration was neither observed with an analogous compound featuring a (R)-2-aminoethyl substituent (19) nor with a weakly basic 2-pyrazine derivative (20). The rate-enhancing effect of methylene-linked amino groups has been described previously for propiolamide- and acrylamide-derived EGFR inhibitors.(94,95) In the suggested mechanism, the basic amine increases the nucleophilicity of the thiol group by hydrogen bonding/deprotonation (Figure 6B). The distinct effect of the (R)-pyrrolidine residue compared to the primary propargylic amine in 19 might be rationalized by the spatial requirements for proton abstraction. An X-ray crystal structure confirmed covalent binding of compound 17 to Cys803 in ErbB4 (Figure 7) while EGFR labeling at Cys797 was proven by MS. Key compound 18, a potent EGFR, ErbB2, and ErbB4 inhibitor (IC50 = 7, 13, and 66 nM, respectively) with cellular IC50 values in the midnanomolar range featured >100-fold selectivity against 30 from 31 kinases in a small panel. Remarkably, it did not inhibit interleukin-2-inducible T-cell kinase (ITK) possessing an equivalently positioned cysteine. Dosed orally at 30 and 100 mg/kg b.i.d., compound 18 was an effective inhibitor of tumor growth in a murine BT474 cell xenograft model while compound 19 showed slightly lower activity in the same model system.
Figure 6
Figure 6. 6-Ethynylthienopyrimidine as covalent ErbB kinase inhibitors. (A) 6-Ethynylthieno[3,2-d]pyrimidine and 6-ethynylthieno[2,3-d]pyrimidine-derived inhibitors. (B) Suggested mechanism of cysteine addition.
Figure 7
Figure 7. X-ray crystal structure of compound 17 in complex with the ErbB4 kinase domain (PDB 2R4B). The terminal alkyne moiety has reacted with Cys803 to form a vinyl thioether adduct. The N1-atom of the pyrimidine ring is further anchored to the backbone NH of Met799 in the hinge region via a hydrogen bond. The pyrimidine N3-atom is engaged in a water-mediated hydrogen bond to the side chain of Thr860 preceding the conserved DFG motif.
In several follow-up studies, the same team investigated the effects of substituents at the 4-position of the pyrrolidine ring,(96,97) the stereochemistry(97) at the pyrrolidine substituent, and modifications at the aniline headgroup.(98) Although some improvements of ADMET properties were achieved, no major advances in terms of biochemical and cellular potency and covalent labeling efficiency could be made. Moreover, it should be pointed out that neither biochemical nor cellular inhibitory potencies showed a clear correlation with the propensity to covalently modify the target in vitro, accentuating that the comparatively slow cysteine addition was not the key driver of biological activity. Since no further studies with this inhibitor class have been reported since 2009, it can be assumed that their development was discontinued. Nevertheless, the provided data shows that heteroarene-derived Michael-type acceptors are compatible with a physiological environment and can in principle be used as CRGs in drug discovery. Systematic studies, however, will be required to explore the tunability and versatility of this type of warhead. Moreover, when alkynylated heteroaryl groups are employed as acceptors, the stability of the adducts toward a second thioether addition and accompanying thiol exchange reactions (see section 2.3) will necessitate further investigation.
More recently, the group of Rob Leurs modified reversible 2-aminopyrimidine-based antagonists of the human histamine H4 receptor with a 2-vinyl substituent to covalently address Cys98 located in the transmembrane domain (TM) 3.(99) Both VUF14480 (22, Figure 8), the key compound of this study, and its nonelectrophilic analogue VUF14481 (23), bound the hH4 receptor with similar (apparent) affinities in the upper nanomolar range. Both compounds acted as partial agonists and potencies in a GTPγS-binding assay were comparable to the respective affinities but time-dependence was not determined in either assay system. As expected, 22 reacted with GSH and cysteine ethyl ester to form covalent adducts, while 23 was unreactive. Unfortunately, only indirect evidence for the covalent engagement of Cys98 was provided and no validation by MS or X-ray crystallography performed. To this end, the authors showed that only 22, but not 23, decreased [H3]-histamine binding after washout, an effect that was not observed when performing the same experiment with the hH4 C98S mutant. However, even at the highest concentration of 10 μM (incubation for 1 h), 22 only partially blocked [H3]-histamine binding after washout, indicating a slow reaction and incomplete covalent labeling. Interestingly, the affinities of both compounds did not significantly differ between the wild-type receptor and the C98S mutant, suggesting reversible binding to be the main contributor to the latter.
Figure 8
Figure 8. 2-Vinylpyrimidine-derived H4 receptor ligand VUF14480 and the unreactive analogue VUF14481.
2.5. Cysteine Addition to Nonactivated Terminal Alkynes
Propargylamines such as selegiline and rasagiline are well-known as mechanism-based covalent monoamine oxidase (MAO)-B inhibitors in the treatment of Parkinson’s disease,(100,101) and terminal alkynes have further found application as activity-based probes for CYP enzymes.(102,103) In these compounds, however, the respective alkynes undergo oxidative activation to generate electrophiles, which can covalently engage their protein targets. While strain activated alkynes are known to react with cysteine residues,(104,105) the nonactivated terminal alkynes employed in CuAAC click reactions(106) have been assumed to be inert in biological samples. In this light, recent reports on protein-templated cysteine addition to nonactivated, and thus poorly electrophilic, terminal alkynes are quite remarkable (exemplified by general structure 24, Figure 9A). In 2013, the groups of Huib Ovaa(107) and Henning Mootz(108) independently discovered this reactivity while employing propargylamide-labeled ubiquitin (Ub), SUMO, or peptides derived thereof, intended for click-chemistry-based labeling. The active site cysteine of deubiquitinating isopeptidases (DUBs) and SUMO proteases selectively attacked the triple bond at the 2-position forming the Markovnikov vinyl thioether adduct. Interestingly, N-but-3-ynyl amide (24a) and even N-hex-5-ynyl amide homologues (24b) were also reactive while the allylic amide 24c showed poor reactivity (Figure 9B). Introduction of a terminal methyl group (24d) or geminal 2,2-dimethyl substitution (24e) at the propargyl residue prevented cysteine addition.(107) The approach could also be applied to other cysteine proteases such as caspase-1(107) or more recently the viral leader protease Lbpro.(109) Although these results partially question the biological inertness of terminal alkynes used in click-chemistry labeling applications, the underlying reaction seems to be quite specific and, so far, limited to certain cysteine proteases. It has been proposed that this reaction requires the stabilization of the intermediary carbanion by the hydrogen bond donors in the oxyanion hole.(110) In the SUMO-protease Senp1, however, mutation of key residues in the catalytic triad (i.e., H533A and D550A) and the oxyanion hole (Q597A) did not prevent the formation of the covalent adduct.(108) Further mechanistic investigations will be required to elucidate the structural requirements promoting these reactions and it remains to be seen, if noncatalytic cysteines could covalently trap propargylamides and similar terminal alkynes in certain settings.
Figure 9
Figure 9. Nonactivated terminal alkynes as cysteine traps. (A) Reaction of C-terminally propargylated ubiquitin 24 with the active site cysteine in DUBs. (B) Reactive (top) and nonreactive (bottom) analogues.
2.6. Cysteine Targeting by Nucleophilic Aromatic Substitution (SNAr) Reactions
Although nucleophilic aromatic substitution (SNAr) reactions have a long history in covalent protein targeting,(111) they are still largely underdeveloped in the field of TCIs. In this type of reaction, covalent labeling is effected by a nucleophile, e.g., a cysteine thiol(ate), displacing a leaving group from an electron-deficient aryl ring. Classically, SNAr reactions have been considered to proceed via a stepwise addition–elimination mechanism (aryne, radical and SN1-type mechanisms are not considered here) involving negatively charged intermediates termed Meisenheimer- or σ-complexes, which are stabilized by the electron-withdrawing group(s) (Figure 10A).(112,113) More recent studies suggest that many SNAr-reactions rather proceed in a concerted manner via Meisenheimer-like transition states.(114) The reactivity of (hetero)arenes in SNAr reactions increases with the electron deficiency of the aromatic system. Reactivity can be readily increased by adding electron-withdrawing (typically −M) substituents or heteroatoms, which are ideally positioned to the ortho- or para-position of the leaving group. In most cases, the rate of SNAr-reactions is determined by the attack of the nucleophile while elimination of the leaving group is faster.(113) Therefore, the SNAr reactivity of aryl halides typically ranks F ≫ Cl ≈ Br > I, roughly correlating with the increasing polarization of the Cδ+–Xδ− bond. However, different rankings might be observed because the loss of the nucleofuge can also become rate limiting under certain conditions. A detailed discussion of all factors influencing SNAr reactions is beyond the scope of this article and can be found elsewhere.(113) Yet, it is important to note that the reactivity of SNAr electrophiles can be readily adjusted by varying the electronic nature of substituents, and the leaving group.
Figure 10
Figure 10. SNAr-based covalent ligands. (A) Classical mechanism of the SNAr reaction with cystein. (B) Selected examples of early SNAr-based ligands and reagents. Leaving groups are highlighted in red.
In early applications, SNAr warheads have been exploited in inhibitors like the 2-chloroquinoxaline L-764406 (25, Figure 10B),(115) the 2-chlorobenzamide GW9662 (26)(116) or the 2-sulfonylpyridine GSK3787 (27),(117) which target peroxisome proliferator-activated receptors (PPARs) by labeling different noncatalytic cysteines. 2-Sulfonyloxadiazoles, as exemplified by 28, have been used to covalently block the catalytic serine of AmpC β-lactamases(118) or as maleimide replacements for the generation of serum-stable antibody conjugates.(119) Other heteroarylsulfone-derived SNAr reagents such as MSBT (2-(methanesulfonyl)benzothiazole, 29)(120) have been employed as cysteine capping reagents and for protein conjugation.(121) The SNAr reactivities of perfluoroarenes(122,123) and dichloro-s-tetrazine(124) have been further exploited for peptide stapling. Notably, the cysteine reactivity of highly activated SNAr electrophiles has been correlated with their skin sensitization potential.(125)
In 2011, a team of researchers around Kiplin Guy identified methylsulfonyl nitrobenzoates exemplified by MLS000389544 (30) as low micromolar irreversible inhibitors of the thyroid hormone receptor (TR) β/steroid coactivator (SRC) 2 interaction by high throughput screening (HTS) of a library of 500000 compounds.(126) These compounds selectively labeled Cys298 as determined by MS, the same cysteine that had previously been shown to react with Michael acceptors formed in situ from β-aminoketones.(127) It is noteworthy that nucleophilic displacement of the methylsulfonyl residue was selective in the proximity of an activated ester moiety as an alternative reaction site. As one would expect, removal of the nitro group resulting in a less electrophilic analogue was detrimental to activity. However, the replacement of the methylsulfonyl group by chloride or fluoride also furnished poorly active compounds, which is more surprising because sulfones are typically less reactive than fluorides in SNAr reactions.(113) This finding might reflect the involvement of the methylsulfonyl residue in reversible binding or point to the complex interplay of different factors on SNAr reactivity.
In 2015, John Kuriyan and co-workers reported the sulfonyltetrazole 31 (Figure 11), the NBD-dye 32, and the sulfonylpyrimidine carboxamide 33 as low molecular weight inhibitors antagonizing the interaction between the tandem Src homology (SH) 2 domains of the nonreceptor tyrosine kinases ZAP-70 and Syk and doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs).(128,129) These compounds, which were identified by HTS, inhibited the SH2–ITAM interaction at low micromolar concentrations in a time-dependent manner. X-ray and MS experiments confirmed the specific binding to single cysteines when no excess of the reagents was used. The X-ray structures of compounds 31 (PDB 4XZ0) and 32 (PDB 4XZ1) in complex with the ZAP-tSH2 domain did not show any specific interactions with the protein and switching from TCEP- to DTT-containing buffer completely abolished inhibition by all three compounds. Therefore, it can be assumed that the high intrinsic reactivity of these electrophiles precludes application in cells or in vivo.
Figure 11
Figure 11. Covalent inhibitors antagonizing the interaction of ZAP-70 and Syk with ITAMs. Leaving groups are highlighted in red.
A reactivity analysis of electron-deficient halogenated (hetero)aryl fragments in cellular proteomes was reported in 2014 by Eranthie Weerapana and co-workers.(130) By using an alkyne-tagged series of chloronitrobenzoic amides (general structure 34, Figure 12A), they found that selectivity critically depends on the positioning of the nitro group. As expected, a meta relationship between the nitro moiety and chloride leaving group gave an unreactive compound, whereas compound 34a (Figure 12B) with the nitro group in the para-position was the most reactive. Dichloropyridines and -pyrimidines (general structure 35) also showed limited reactivity, while an analogous triazine (35a) caused more promiscuous labeling reflecting the highly electron-deficient nature of this heteroarene. Intriguingly, however, 34a and 35a affected distinct sets of proteins, and peptide mapping showed that 34a preferentially labeled cysteine thiols while 35a had an unexpected preference for the lysine side chain. Although this behavior could not be fully rationalized, complementary experiments showed that 35a preferentially reacts with cysteine in solution, emphasizing that solution reactivity is not necessarily predictive of reactivity within protein binding sites.
Figure 12
Figure 12. Electron-deficient (hetero)aryl probes used for evaluation of SNAr-based labeling in proteomes. (A) General structures. (B) Selected compounds preferably labeling cysteine or lysine.
A recent study from Walter Fast and co-workers investigated 4-halopyridines as quiescent SNAr electrophiles.(131) They found protonation of the pyridine nitrogen atom to be a critical factor for the reactivity of this compound class. To this end, the reaction with GSH at physiological pH was compared between 4-chloropyridine (36, Figure 13A) and its charged N-methylated analogue (37, Figure 13B). Earlier data from Flanagan et al. had already demonstrated that 2-chloropyridine reacts only slowly with GSH at pH 7.4, with a rate constant in the same range as observed for N-methylacrylamide.(12) Similarly, 36 showed very low reactivity comparable to that of ampicillin, styrene oxide, and acrylamide. In contrast, the N-methylated analogue’s reactivity increased by more than 3 orders of magnitude, thus being in the same range as the one of iodoacetamide. Arguing with the classical SNAr mechanism, the accelerating effect would be attributed to better stabilization of the intermediate adduct being a neutral dihydropyridine instead of a negatively charged σ-complex in this case (Figure 13B). Against the initial expectation that 4-chloropyridine would be more reactive than the respective bromo and iodo-derivative, reaction rates with thiophenol increased in the order Br > I > Cl (4-fluoropyridines were not included in this study). The same ranking was obtained when the 4-halopyridines were assayed for their capability of inactivating dimethylarginine dimethylaminohydrolase (DDAH) 1 by reacting with the catalytic cysteine residue. For further investigations, a series of disubstituted 4-halopyridines with various substituents at ortho- or meta-position was prepared (general structure 38, Figure 13C). All of these compounds showed no or little reactivity in the GSH assay, even if an electron-withdrawing substituent was present in the C3-position. Profiling this structurally heterogeneous series against DDAH revealed no clear correlation between kinact/KI and the substitution pattern. However, in a set of 2-methylpyridines with an increasing number of fluorine substituents at the methyl group (pKa values ranging from 4.7 to 0.4), a correlation between the pKa of the protonated pyridine and reactivity was observed. Counterintuitively, more electron-deficient compounds reacted more slowly. This suggests that the DDAH enzyme, which possesses a proximal aspartate (Asp66), would stabilize the protonated pyridine, thereby enhancing its reactivity. However, no X-ray crystal structures or mutation studies were provided to fortify this hypothesis. Further experiments were performed with alkyne-tagged probes 39 and 40 (Figure 13D) in soluble Escherichia coli proteomes. Gel-based analysis showed that 39 only modifies a few proteins and the number even decreases when cell lysates are denatured prior to exposure. In contrast, N-methylated analogue 40 labeled much more proteins and the number increased substantially in denatured lysates. These observations suggest that the neutral 4-halopyridines require activation by the protein environment for becoming reactive, while the charged N-methyl-4-halopyridines can promiscuously label surface-exposed nucleophiles, which are more abundant in the denatured samples.
Figure 13
Figure 13. 4-Halopyridines as quiescent SNAr electrophiles. (A) SNAr-reaction with 4-chloropyridine according to the classical mechanism. An anionic Meisenheimer intermediate is formed. (B) Analogous mechanism of the reaction with N-methyl-4-chloropyridine. A neutral dihydropyridine species is formed as the intermediate. (C) General structure of the investigated compounds. (D) Alkyne-tagged probes used for proteomic analysis.
In a recently published drug discovery campaign, Robin Fairhurst and co-workers from Novartis aimed to identify covalent inhibitors targeting a rare cysteine (Cys552)(35) located in the hinge region of the fibroblast growth factor receptor (FGFR) 4 tyrosine kinase.(132) A high throughput screen revealed compound 41 (Figure 14A) as a nanomolar inhibitor (IC50 = 32 nM) of wild-type FGFR4 sparing the FGFR4 C552A mutant. The compound did not hit the other FGFR family members (FGFR1–3), which are devoid of this cysteine. The high activity of this structurally simple molecule can be attributed to displacement of the 6-pyridyl chlorine atom by the cysteine’s thiol. Covalent modification at Cys552 was confirmed by MS and X-ray crystallography (Figure 14B). The compound possessed a 170-fold lower rate constant (kinact/KI = 3.0 × 104 M–1s–1) than an acrylamide-based inhibitor from the same study targeting Cys477 common to all FGFR family members. Although 41 selectively labeled Cys552 under the conditions used in MS and X-ray experiments, a 10-fold excess also modified Cys477 indicating at least some promiscuity. Interestingly, the latter modification seems to leave kinase activity unaffected, highlighting the fact that enzymatic or competition-based kinase assays are typically not suited for the identification of nonactive site binders. Although the authors recognized the potential of this compound for optimization in terms of reactivity, potency, and selectivity, the inhibitor was dropped in favor of a covalent-reversibly binding aldehyde (vide infra, section 2.12) owing to the short FGFR4 resynthesis half-life (<2 h).
Figure 14
Figure 14. (A) Structure of the covalent FGFR4 inhibitor 41. (B) X-ray cystal structure of 41 in complex with the FGFR4 kinase domain (PDB 5NUD). Both pyridine rings form a hydrogen bond with the backbone NH group of Ala553, while the nitro group stabilizes the active conformation via an intramolecular hydrogen bond with the diarylamino NH. The covalent bond with Cys552 is formed by SNAr displacement of the 6-chloro group from the 2-amino-3-nitropyridine moiety. A weak water mediated H-bond between the nitro substituent and the Arg483 guanidinium group was omitted for clarity.
Weijie Hou and co-workers reported in early 2018 on putatively covalent EGFR inhibitors featuring SNAr warheads to address Cys797.(133) Their rationale was the replacement of the α,β-unsaturated amide found in afatinib and analogous compounds by an electron-deficient aryl ring equipped with a halide leaving group (general structure 42, Figure 15). Several potent inhibitors with low nanomolar affinities were identified, but activity differences between electrophilic and nonelectrophilic compounds were only moderate. Covalent modification was not directly assessed, but washout experiments with key compound 42a indicated only reversible binding although the inhibitor reacted with GSH confirming its inherent reactivity. As the EGFR is known to react with various acrylamide-derived inhibitors,(134) it can be assumed that inappropriate positioning of the electrophile prevents covalent trapping in this case.
Figure 15
Figure 15. EGFR inhibitors with SNAr warheads which do not form the predicted covalent bond with Cys797.
Although the above studies show that targeted covalent inhibition with SNAr warheads is feasible, no substantial efforts on the (successful) compound optimization were reported nor were the compounds profiled in a broader array of assays. A more comprehensive medicinal chemistry study on verifiable SNAr-based irreversible inhibitors was published by Kevin Chen and colleagues at Merck Sharp & Dohme.(135) The researchers optimized a screening hit based on an indole core (43, Figure 16) to inhibit the hepatitis C virus (HCV) NS5B polymerase. While optimizing the indole N1-substituent, the introduction of a nitrile group ortho to the fluoride at a 2-fluoro-5-methylsulfonyl benzyl residue (45) was serendipitously found to promote a 100-fold improvement in activity in a cellular replicon assay. As the parent compound 44 was known to bind to the “palm” site of the NS5B protein, covalent bond formation with the noncatalytic Cys366, a residue that had previously been targeted with benzylidene rhodanine-derived covalent-reversible inhibitors,(136) was assumed to be responsible for this unexpected leap in activity. A variety of analogues with different electron-withdrawing substituents and leaving groups at the phenyl ring was prepared, but no electron-deficient heteroarenes had been explored at that stage. While most modifications gave only weak to moderately active compounds, a strongly electron-withdrawing nitro group in the para-position of the halide leaving group (46 and 47) was able to promote excellent biochemical and cellular activity. Notably, binding kinetics were not investigated. Further optimization yielded key compound 48, which exhibited a good PK profile in monkeys and dogs. The inhibitor featured an oral bioavailability of 30% and 95%, an AUC (po, 3 mg/kg) of 3.4 and 11 μM·h, and a half-life of 1.3 and 1.2 h, respectively, in the aforementioned species. The X-ray crystal structure of 48 in complex with the HCV NS5B polymerase (PDB 3TYQ) as well as MS experiments unambiguously confirmed the expected covalent binding mode. An NMR-based method (ALARM-NMR)(137) exploiting the high nucleophilicity of cysteines in human La antigen showed no promiscuous thiol reactivity. Moreover, the compound was found to be clean in kinase and protease panels. Although the nitro group was regarded with skepticism due to known potential safety issues caused by partial reduction products,(138) no toxicity could be observed in a Salmonella/mammalian microsome mini-AMES assay, an in vitro micronucleus assay and in single-dose animal PK studies.
Figure 16
Figure 16. Development of covalent HCV NS5B polymerase inhibitors with SNAr warheads.
In a follow-up study, the same team evaluated heterocyclic substitutes for the 2-fluoro-5-nitrobenzyl residue.(139) Although the nitro group did not promote any obvious toxicity in the above study, it was considered to be safer to avoid this functionality. An initial meta-linked 2-chloropyridine series exemplified by 49 (Figure 17A) gave acceptable potencies in the biochemical assay, however, cellular EC50 values increased to the low micromolar range. As it was shown later, these moieties were not capable of covalent target engagement. Replacement of the pyridine ring by a 2-chloroquinoline moiety rescued cellular activity. Although small substituents in the C6- and C7-position of the quinoline ring were tolerated, the best results were obtained with an undecorated 2-chloroquinoline moiety. Key compound 50 featured low nanomolar activities in both assays. Covalent binding to Cys366 was proven by MS and X-ray crystallography (Figure 17B), and no labeling of any of the enzyme’s other 21 cysteines was detected. Intriguingly, the compound was stable toward harsh chemical conditions such as refluxing in aqueous lithium hydroxide or 0.5 M ammonia/dioxane pointing out the low intrinsic reactivity of the warhead. The compound possessed a similar clean profile as 48, showed no significant CYP or hERG interactions, only limited toxicity up to 2000 mg/kg in rats, and favorable in vivo drug metabolism and pharmacokinetic (DMPK) properties in rat, dog, and monkey. Compound 50 was thus suggested as a candidate for further development.
Figure 17
Figure 17. Second generation NS5B polymerase inhibitors with SNAr warheads. (A) Reversibly binding 2-chloropyridine 49 and the irreversibly binding quinoline analogue 50. (B) X-ray crystal structure of key compound 50 covalently bound to Cys366 of HCV NS5B polymerase (PDB 4MZ4). The compound is further anchored by hydrogen bonds between the 2-pyridone moiety and the backbone carbonyl atom of Gln446 and the NH group of Tyr448. The carboxylate and the quinoline N1-atom are linked to different residues via water-mediated hydrogen bond networks (the second sphere of water molecules and beyond was omitted for clarity). A second conformation of the Cys366 was omitted as well.
In summary, the discussed reports point out the high potential of SNAr chemistry in TCI discovery. Although only few medicinal chemistry programs have been published and little systematic investigation has been performed, the favorable properties of SNAr warheads such as tunability, synthetic accessibility, and structural rigidity will probably stimulate further exploration of this structure class. Moreover, electron-deficient (hetero)arenes are already present in many known ligands and approved drugs with excellent ADME properties. If a properly positioned nucleophile is present in the binding site, such a ligand might be readily turned into an irreversible modifier simply by the installation of suitable leaving groups. Besides the efforts discussed above, it is worth mentioning that an elegant approach to target DNA cytosine-5-methyltransferases covalently via an SNAr mechanism has very recently been reported in two studies by Akira Matsuda, Satoshi Ichikawa, and co-workers.(140,141) However, their approach addressing an active site cysteine via a suicide mechanism is beyond the scope of this Perspective. The interested reader is therefore referred to the original articles.
2.7. Cysteine Alkylation by Strain Release Reagents
A very elegant method for selective cysteine labeling by using strain release reagents was described in 2016 by the group of Phil Baran.(142,143) Although the strain-promoted addition of thiols to [1,1,1]-propellane derivatives has been known since the 1980s,(144) this reaction has not been applied to cysteine conjugation until recently. While [1,1,1]-propellane was found to be a useful building block for the derivatization (“propellerization”) of alkylamines by means of “turbo-Grignard” reagents,(145) phenylsulfonyl bicyclobutanes (e.g., compound 51, Figure 18) proved to be suitable for cysteine labeling applications. These bench-stable reagents selectively cyclobutylated cysteine in glutathione and in highly functionalized peptides under basic conditions while leaving other nucleophilic amino acid side chains untouched (Figure 18).(142) Analogous cyclopentylation reactions were carried out even in a stereospecific manner by using chiral 1-(phenylsulfonyl)bicyclo[2.1.0]pentanes (housanes, e.g., compound (+)-52) as the strain-release reagent.(143) The reactivity of the strained cycles could be readily tuned by modulating the electronic properties of the aryl system linked to the sulfonyl group. As expected, the attachment of electron-withdrawing substituents increased reaction rates while electron-donating substituents had the opposite effect. The adducts were chemically stable even in the presence of TCEP, but stability studies in a more physiologically relevant setting remain elusive. In a GSH reactivity assay, previously reported by researchers from Pfizer (vide infra),(12) strain-release warheads were benchmarked against common α,β-unsaturated amides and vinyl sulfonamides (see Table 1 for a representative selection). Half-lives spanned from 4 h for the 3,5-difluoro derivative (no analogues with stronger electron-withdrawing groups have been evaluated) to 19 h for the 4-methoxy analogue. The compounds thus proved to be significantly less reactive than the common N-phenylacrylamide motif (t1/2 = 0.9 h) or N-(vinylsulfonyl)pyrrolidine (t1/2 = 0.53 h) but in the same range as N-benzyl acrylamide (t1/2 = 15 h).
Figure 18
Figure 18. Strain-release reagents and their reaction with a cysteine-containing peptide.
Table 1. Influence of the Aryl Substituent on the Reactivity of Phenylsulfonyl Bicyclobutanes in a GSH Stability Assay and Comparison with Common Acrylamides
Due to their well-balanced and tunable reactivity toward cysteines along with the limited spatial requirements and the defined geometry, these electrophiles might be well-suited as warheads in drug discovery. Moreover, the replacement of the aryl sulfone moiety by analogous sulfonamides, carboxamides, esters, or ketones or the attachment of substituents to the strained ring system may enable the modulation of reactivity over a wider range. Unfortunately, no studies demonstrating the applicability of strain-release warheads in a complex biological setting have been published so far. One potential issue with this CRG is the underlying synthetic chemistry. Although Baran and co-workers have developed an operationally simple protocol for the preparation of the strain-release reagents discussed above, the flexible implementation of the latter into chemically more complex settings may require optimization and profound chemical expertise.
2.8. Cysteine Alkylation by Nucleophilic Displacement of Alkyl Halides
Although aryl halides are abundant in approved and investigational drugs, their alkyl counterparts are rare, with the notable exception of DNA alkylating agents(146) and unreactive alkyl fluoride motifs, that have been frequently applied in drug discovery, e.g., as bioisosteres or for modulating physicochemical properties.(147,148) The most representative sp3 halides used to covalently modify biological targets are α-halomethylketones and the analogous esters and amides. α-Halomethylketones have a long history as protease probes where the keto group is attacked by the active site nucleophile while the proximal alkyl halide irreversibly labels the enzyme, for example, by reacting with a histidine of the catalytic triad.(7) Mometasone, an approved corticosteroid, also contains an α-chloromethylketone residue. Fluoroalkyl groups can be found as the reactive moieties in mechanism-based inhibitors such as eflornithine(149) or trifluoromethyl deoxyuridine derivatives.(150) Moreover, fluoromethylketone (FMK)-tagged adenine analogues were among the first rationally designed covalent kinase inhibitors.(151,152)
The nucleophilic substitution reactions discussed here proceed via an SN2-mechanism (Figure 19A), in which the reaction rate depends on the nucleophile, the substrate, and the leaving group. SN1 reactions, where the rate depends solely on the loss of the nucleofuge to generate a highly reactive carbocation, are not utile in the context of TCI design. Nucleophilic substitutions of nonactivated linear haloalkanes are relatively slow, and increasing steric bulk around the electrophilic carbon center further decreases reactivity. Benzyl or allyl halides are more reactive because the transition state of the SN2-reaction (but also the carbocation in the case of an SN1-mechanism) is stabilized via conjugation with the π-system. A tremendous increase in reactivity is typically observed for α-halomethylketones, which may be rationalized by a further stabilization of the SN2-transition state via conjugation, or by the dual attraction model (Figure 19B).(153) However, the relative increase in reaction rates depends on the nucleophile and is typically more pronounced when strong/negatively charged nucleophiles are employed.(154) Nucleophilic substitution of α-halomethylcarbonyl compounds exclusively proceeds via an SN2-mechanism even if good leaving groups are employed, which can be attributed to the electron-withdrawing carbonyl group destabilizing the intermediate carbocation in SN1 reactions. Reactivity of α-halomethylcarbonyl compounds decreases if +M substituents are attached to the carbonyl group. Consequently, α-halogenated esters are less reactive than the corresponding ketones and the respective amides possess an even lower reactivity.(153) On the other hand, reactivity rises with increasing leaving group properties of the halogen (I > Br > Cl > F). α-Haloacetamides are quite versatile and can be used to label various nucleophiles (typically Cys but also Lys, His, Tyr, activated Ser, and Thr etc.). Iodoacetamides, for example, are highly reactive and known as cysteine capping reagents.(155) Iodoacetamide-derived clickable probes have recently been used to identify hyper-reactive cysteines by means of chemical proteomics.(156) In contrast, α-bromoacetamides, which feature a slightly lower reactivity,(12) are far less common. α-Chloroacetamides, which are the most frequently employed haloacetamides in covalent ligand design, possess further reduced reactivities. Their stability against GSH at pH 7.4 is in the same range as the one of α,β-unsaturated amides,(12) and a linear chloroacetamide-derived alkyne probe showed only moderate levels of labeling in soluble mouse liver proteomes.(157) In the case of aniline-derived α-haloacetamides, GSH reaction rates correlate with the Hammett parameter of the aryl substituent, i.e., electron-donating substituents decrease reactivity and vice versa.(158) Reactivity of α-haloacetamides can be further decreased by adding steric bulk in proximity to the reaction site. Therefore, the reaction rates of α-bromopropionamides are lower than the ones of α-chloroacetamides and α-chloropropionamides are even significantly less reactive than for example N-benzyl acrylamide (Figure 19C).(12) In support of the low and specific reactivity of α-chloropropionamides, a recent ABPP-based study identified (S)-CW3554 (53, Figure 19D) as an irreversible inhibitor of the protein disulfide isomerase A1 (PDIA1) with good selectivity in HEK293 cells.(159) Interestingly, the analogous (R)-α-chloropropionamide specifically labeled a distinct protein, the aldehyde dehydrogenase ALDH2, highlighting the role of the stereochemistry at the α-position of the warhead. However, it should be noted that both enzymes contain strongly nucleophilic active site cysteines and the potential of α-chloropropionamides in TCI design remains to be demonstrated.(160,161)
Figure 19
Figure 19. Alkyl halides as CRGs (A) General mechanism of the SN2 reaction. (B) Dual attraction model rationalizing the enhanced reactivity of α-halocarbonyl compounds. (C) Reactivities of α-halopropion- and acetamides in a GSH assay. Half-lives were determined in the presence of 10 mM GSH at pH 7.4 and 37 °Ca or 60 °Cb. N-Phenylacrylamide is shown for comparison. (D) 2-Chloropropionamide (S)-53, a covalent PDIA1 inhibitor.
As an example of cysteine targeted alkyl halides devoid of an α-carbonyl group, 1,4-disubstituted 5-chloromethyl-1,2,3-triazoles have recently been identified by Alexander Adibekian and co-workers as inhibitors of the O6-methylguanine-DNA-methyltransferase (MGMT), a noncatalytic DNA repair protein that possesses an active site cysteine.(162) The approach was inspired by N-heterocyclic carbene ligands, in which the reactive metal center is shielded by bulky substituents. Analogously, it was hypothesized that the reactivity of the 5-chloromethyl substituent can be modulated sterically by substituents at the N1- and the C4-position of the triazole ring. Ideally, these substituents would at the same time confer selectivity for a given target. A promiscuous clickable probe (54) was first synthesized and reacted with the proteome of MCF7 cancer cell lysates. MGMT was identified as one among the multiple labeled proteins. A small library of triazoles was synthesized by uncatalyzed or ruthenium-promoted azide–alkyne cycloaddition(163) and analyzed in a competition assay against probe 54. In this screening, analogue AA-CW236 (55) was identified as a highly potent MGMT inhibitor (KI = 24 nM), albeit with slow inactivation kinetics (kinact = 0.03 min–1) (Figure 20). The compound did neither cross-react with any of the targets of probe 54 at a concentration of 300 nM nor label the MGMT C145A mutant. Furthermore, MS-based proteomics indicated a clean selectivity profile. The compound, in combination with the DNA alkylation agent temozolomide, significantly increased the O6-alkylguanine levels in MCF7 cells compared to temozolomide alone, indicating a sensitization to the chemotherapeutic drug. Because of the low inactivation rates, however, it is not unlikely that the bulk of the observed effects are promoted by the potent reversible interaction.
Figure 20
Figure 20. 5-Chloromethyl-1,2,3-triazoles as covalent MGMT inhibitors.
Overall, alkyl halides, especially α-haloacetamides, seem well suited as warheads for noncatalytic cysteine residues. Key to the modulation of their reactivity is the nature of the leaving group and the steric environment, especially the substituent at the α-position, which needs to be tolerated by the binding site. Replacing halogens with alternative and tunable leaving groups such as (activated) esters or sulfonates may offer further options to achieve optimally balanced reactivity.
2.9. Cysteine Alkylation by Epoxides and Other Three-Membered Heterocycles
Epoxides (oxiranes) have a long history as warheads to target different types of proteases(7,164) and glycosidases.(165,166) In contrast, other three-membered heterocycles such as aziridines(167) and thiiranes(168,169) have received less attention. The reactivity of these heterocycles arises from their ring strain. Epoxides react with nucleophiles via SN2 mechanisms and in the absence of an acid catalyst, nucleophilic attack preferably occurs at the sterically less hindered position. Epoxides are used as CRGs in approved drugs, e.g., the epoxyketone-based proteasome inhibitor carfilzomib (56, Figure 21)(170) or the antibiotic fosfomycin (57).(171) Fosfomycin is a covalent modifier of bacterial UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) targeting a nonessential cysteine (Cys115 in E. coli).(172,173) Despite being structurally simple, this orally available drug can be safely administered in multigram doses, illustrating the tolerability of epoxide-derived compounds.(171) Moreover, epoxides can behave unreactive even if a proximal cysteine is present in the target’s binding site (e.g., trapoxin A in complex with HDAC 8(174)) and some approved drugs, e.g., ixabepilone (58),(175) feature a bystander epoxide, suggesting that this structural motif cannot be considered a safety issue per se. Styrene oxide has recently been shown to possess a similarly low reactivity as acrylamide or ampicillin toward GSH.(131) On a proteomic scale, terminal alkyl and spiro-epoxides have shown little or no protein labeling.(157) However, despite these facts, epoxides are often regarded with skepticism by medicinal chemists, which probably traces back to the well-known toxic effects of certain epoxy-metabolites as well as their DNA alkylating potential.(6) Despite the wealth of published literature, systematic studies on the factors determining the reactivity of epoxides and other three-membered heterocycles in a complex biological environment remain sparse. Another reason for the low abundance of epoxides in TCI discovery campaigns is their facile hydrolysis by epoxide hydrolases, and factors determining metabolic stability of this compound class are not fully understood. On the other hand, this liability might be used to confer kinetic selectivity in a similar manner as the fumaric acid esters discussed in section 2.1 do. Moreover, the small size and the possibility of being attacked at two proximal positions make epoxides quite versatile CRGs. Two examples for the application of epoxides in the rational targeting of noncatalytic cysteine residues are highlighted below.
Figure 21
Figure 21. Examples of epoxide-containing drugs.
A publication from 2010 by Alessio Lodola and co-workers describes a series of α-acyl epoxides as inhibitors of wild-type EGFR.(176) Notably, other electrophiles such as activated phenyl esters, carbamates, nitriles, and heterocycles containing an electrophilic sulfur atom were also evaluated in this study. Starting from the covalent inhibitor PD168393 (59, Figure 22), replacement of the acrylamide moiety by three different epoxide-containing residues furnished highly potent compounds (IC50 = 0.5, 0.5, and 1.2 nM for 60a, 60b, and 61, respectively) with cellular activities in the low nanomolar range. In contrast, the β-naphthylamine-derived control compound 62 did not show any activity in the A431 cell assay, suggesting the absence of general cytotoxicity. In an LC-MS-based GSH binding assay, epoxide 60a did not show any adduct formation after 1 h, while a 36% conversion was found for acrylamide 59. Irreversible binding of the epoxides was suggested by washout experiments, however, no further analysis of binding kinetics was performed and no confirmation of covalent target modification by MS or X-ray crystallography reported. Therefore, it cannot unambiguously be concluded if covalent binding took place. Notably, Daniel Rauh and co-workers observed only very weak activity of the analogous epoxide 63 on the cSRC S345C mutant sharing an equivalently positioned cysteine.(177)
Figure 22
Figure 22. α-Acyl epoxides as warheads for putatively covalent EGFR inhibitors.
In a similar approach from our own group, an epoxide moiety was used to invert the isoform selectivity profile of ruxolitinib (64, Figure 23), an approved JAK1/JAK2/tyrosine kinase (TYK) 2 inhibitor.(178) In search of selective JAK3 inhibitors, molecular modeling proposed that replacing ruxolitinib’s (R)-3-cyclopentyl propanenitrile side chain by a propylene oxide moiety could address JAK3 Cys909, which is unique within the JAK family. We prepared a series of ruxolitinib-derived triazoles and epoxy analogue 65 appeared to be a potent JAK3 inhibitor (IC50 = 35 nM) with a high selectivity (70–160-fold) in the JAK family. Because the other JAK family members possess a serine at the position equivalent to Cys909, the selectivity shift, which was not observed for nonreactive control compounds, suggests a covalent interaction with Cys909. However, covalent modification was not unambiguously proven since we discontinued this series in favor of tricyclic covalent-reversible JAK3 inhibitors.(179)
Figure 23
Figure 23. Ruxolitinib-derived triazoles with a propylene oxide warhead as selective JAK3 inhibitors.
Aziridines have been used for targeting active site carboxylates in glycosidases,(180) but reports on their application as CRGs in chemical biology and drug discovery remain sparse. Their avoidance can probably be traced back to the well-known DNA-alkylating properties of aziridinium ions, which are famous as the active species generated from N-lost derived chemotherapeutics.(181) Nevertheless, a recent example employing an aziridine as TCI warhead was provided by the groups of Kevan Shokat and John Burke.(182) With the aim of covalently addressing the oncogenic K-Ras G12D mutant via the aspartate, different electrophilic head groups were attached to an optimized irreversible ligand of K-Ras G12C (general structure 66, Figure 24). Despite the reactivity of the representative aziridine 66a toward carboxylate groups in solution, negligible modification of K-Ras G12D was observed. In contrast, the oncogenic K-Ras G12C mutant was fully labeled. Binding was independently confirmed by differential scanning calorimetry and by hydrogen–deuterium exchange MS (HDX-MS). X-ray-crystallography and MS finally revealed the exact binding mode (Figure 25). It is worth mentioning that the attack of the thiol group occurred at the sterically more hindered α-carbon atom.
Figure 24
Figure 24. K-Ras G12D or G12C-targeted covalent inhibitors. Key compound 66a features an aziridine warhead.
Figure 25
Figure 25. X-ray crystal structure of the K-Ras G12C mutant covalently bound to compound 66a (PDB 5V6V). Cys12 forms the covalent bond by opening the aziridine ring at the β-position. The indole NH and quinazoline N1-atom are involved in charge-assisted hydrogen bonds to the side chains of of Asp69 and Arg68, respectively, while the piperidine carboxamide oxygen interacts with the side chains of Tyr96 and Asp92 via water-bridged hydrogen bonds.
2.10. Cysteine Targeting by Nitroalkyl Groups As Masked Electrophiles
The covalent complex formation between 3-nitropropionate (3-NP, 67a, Figure 26) and Mycobacterium tuberculosis isocitrate lyase (ICL) was recently investigated by the group of Andrew Murkin. They found this compound to irreversibly inhibit ICL without the requirement for cofactors or prior redox-activation of the nitro group.(183) After 20 h of incubation with 3-NP, only negligible enzyme activity could be recovered by jump dilution, an observation that was not influenced by the addition of DTT. However, the presence of glyoxylate increased reaction rates, suggesting cooperative binding. ESI-MS and X-ray crystallography (PDB 6C4A) revealed the formation of a thiohydroximate by reaction of the inhibitor with Cys191, an active site residue acting as a general base in ICL catalysis.(184) An inverse solvent isotope effect indicated the thiolate form to be the nucleophilic species. The reaction could be rationalized by the CH-acidic nature of 3-NP (pKa = 9.0) generating significant amounts of the conjugate base propionate-3-nitronate (P3N, 67b) at neutral pH. A mechanism was proposed in which Glu285 protonates P3N at an oxygen atom to transform the nucleophilic nitronate into an electrophilic nitronic acid. The latter could react with Cys191 to form the thiohydroximate by water elimination, probably via a second protonation step involving Arg228. The reaction mechanism was supported by kinetic analyses showing that preformed P3N (kinact/KI = 2.6 × 104 M–1 s–1) inactivated the enzyme approximately 100 times faster than 3-NP. Accordingly, nitroalkanes should be suitable as cysteine-targeted warheads for proteins hosting a proper combination of a sufficiently reactive cysteine and proximal acidic residues capable of generating the nitronic acid electrophile. Besides the aforementioned concerns on nitro groups in drug discovery, the generalizability of this concept remains to be demonstrated. To this end, chemoproteomic studies with nitroalkyl-derived probes would provide valuable information on the scope of this reactivity with respect to other target proteins.
Figure 26
Figure 26. 3-Nitropropionate, propionate-3-nitronate, and the suggested reaction mechanism with ICL.
2.11. Reversible Cysteine Addition to α-Cyanoacrylamides
Covalent reversible targeting of noncatalytic cysteines has recently emerged as a strategy to benefit from the advantages of irreversible TCIs (e.g., long residence times and the (potentially) increased selectivity) while avoiding liabilities like permanent off-target modification and the risk of idiosyncratic toxicity.(28) The rational implementation of this concept in TCI design has been pioneered by the group of Jack Taunton, who introduced the β-substituted α-cyanoacrylamide functionality as a promising covalent-reversible warhead for cysteine.(25) Although this electrophile can be considered highly reactive toward thiols, the increased α-CH acidity and thermodynamic destabilization of the β-thioether adduct(185) favor the reverse reaction and thereby dissociation from off-targets or peptides, which are not capable of stabilizing the covalent complex by noncovalent interactions. Interestingly, a recent crystal structure of the kinase JAK3 in complex with the selective α-cyanoacrylamide-based inhibitor FM-409 (68, Figure 27) shows the coexistence of both the covalently bound and the unreacted form (PDB 5LWN), supporting the notion of covalent-reversible binding.(179) By adding steric bulk to the β-position of the α-cyanoacrylamide(11) or by replacing the amide group by an electron-withdrawing heteroarene,(186) the intrinsic reactivity of this CRG and the dissociation rates of the derived covalent-reversible inhibitors can be modulated.(185) More details are provided in a recent Perspective article(40) and subsequent publications highlighting the merit of this warhead class in the design of EGFR-(187) and highly isoform-specific JAK3 inhibitors.(188) Global profiling of the covalent interactions of this chemotype in proteomes, however, is complicated by the reversible nature of protein modification and only a few studies assessing target and off-target binding in cells have been published to date. For example, compound 69 (Figure 27), a low micromolar covalent-reversible inhibitor of glycosaminoglycan (GAG) sulfotransferases(189) was tested for competition with a known alkynylated iodoacetamide probe.(13,156) The compound neither competed with iodoacetamide-labeling in some isolated proteins containing hyper-reactive cysteines(156) nor did it change the labeling profile in the cellular proteome of Neu7 astrocytes, indicating good specificity despite the highly reactive warhead.(189) The compound further showed good stability in rodent and human liver microsomes and moderate in vivo PK properties after intravenous injection in rats. In another study, prolonged cellular target engagement has been demonstrated for α-cyanoacrylamide-derived covalent-reversible BTK inhibitors (e.g., compound 70, Figure 27) in isolated Ramos B cells and in PBMCs after oral dosing in rodents.(11) Notably, further development of this compounds furnished PRN1008,(190−192) a covalent-reversible oral BTK inhibitor of undisclosed structure currently being in phase II/III clinical trials for the treatment of pemphigus (NCT02704429/NCT03762265 on https://www.clinicaltrials.gov) and immune thrombocytopenic purpura (NCT03395210). A dually activated Michael acceptor is also present in the approved catecholamine-O-methyltransferase (COMT) inhibitor entacapone (71).(193) Although further studies are clearly required to determine the biological fate of α-cyanoacrylamides and analogous Michael acceptors, the currently available data suggest them to be promising warheads for in vivo use.
Figure 27
Figure 27. α-Cyanoacrylamide-derived covalent-reversible inhibitors.
Beyond α-cyanoacrylamides, benzylidene rhodanines have been reported as covalent-reversible Michael-type warheads.(136) However, covalent binding does not seem to be a general feature of this compound class.(194) In contrast, arylidene dinitriles are very reactive reversible Michael acceptors, but the reverse reaction seems to be competed by side reactions in this case.(188) Moreover alkynone-derived Michael acceptors (cf. section 2.3)(79) and β-thiomethyl α-cyanovinylsulfones(195) have recently been shown to react reversibly with thiols. According to the mechanism in Figure 5B, however, the reverse reaction is rather a thiol exchange, which might complicate the implementation in TCI design. Nevertheless, the current combinations of electron-withdrawing groups for generating covalent-reversible Michael acceptors with specifically tuned reactivities are by far not exhaustive, leaving room for further optimization. It will be interesting to see if alternative chemotypes such as vinyl sulfones or sulfonamides equipped with an additional electron-withdrawing group in the α-position can further extend the scope (e.g., toward lysines, vide infra) of this covalent-reversible chemistry.
2.12. Reversible Cysteine Addition to Aldehydes
Aldehydes and ketones are common warheads in proteolytic enzyme inhibitors.(7,26,196) They react reversibly by forming tetrahedral hemi(thio)acetals and ketals mimicking the transition state of amide bond cleavage.(197) These moieties, however, are far less commonly employed to address noncatalytic cysteine or lysine residues (some recent examples for lysine targeting by Schiff base formation can be found in refs (198−200)). In a recent drug discovery program by Fairhurst and co-workers, the utility of aldehydes in covalent-reversible kinase targeting has been demonstrated.(132,201) In the screening campaign, which also identified SNAr-based irreversible inhibitor 41 (see section 2.6), a set of closely related 2-formylquinoline amides exemplified by compound 72 (Figure 28, IC50 = 65 nM) was discovered. These compounds were potent FGFR4 inhibitors with good selectivity in the FGFR family but also against the FGFR4 C552A mutant and a panel of over 50 kinases. Replacement of the formyl warhead by a proton, hydroxymethyl, or acid group was detrimental to activity. In accordance with these data, modeling studies suggested covalent-reversible binding to the middle-hinge Cys552 by hemithioacetal formation. The quinoline nitrogen atom and the carboxamide proton were found to be of crucial importance because they stabilize a pseudotricyclic arrangement via intramolecular hydrogen bonding, thereby positioning the aldehyde for covalent interaction. Moreover, the electron-withdrawing ortho-quinoline scaffold further increases the aldehyde’s electrophilicity. Although certain questions concerning toxicity and metabolic stability of the aldehyde moiety remained,(202) this compound class was selected for optimization due to its excellent potency and selectivity profile. Covalent-reversible inhibition was considered more promising than irreversible targeting due to the short resynthesis half-life (<2 h in HUH7 and Hep3B cells) of FGFR4. The 2-formylquinoline amide scaffold, however, was unsuited for further development due to very low solubility. A scaffold morphing approach increasing the sp3 portion led to bioisosteric 2-formylpyridine ureas (exemplified by 73a–d) with improved solubility. Replacement of the trifluoromethyl (R1) group by a nitrile (73b) decreased lipophilicity, and the introduction of an oxygen atom in the saturated ring system (73c) or polar R2 substituents (73d) further increased both solubility and potency. The advanced lead compound 73d featured low nanomolar potency in an enzymatic and a Ba/F3 cell assay (IC50 = 1.3 and 18 nM, respectively). Residence times (pH 7.0) for the slightly less potent compounds 73a–c were in the range between 105 min (73c) and 272 min (73a) and thus much increased compared to an equipotent noncovalent inhibitor (<1.4 min). The significant off-rates (between 3.7 × 10–3 and 9.6 × 10–3 min–1) pinpoint the highly reversible nature of hemithioacetal formation.(203) Consistently, no covalent adducts were found in mass-spectrometric experiments. Although no inhibitor-bound X-ray crystal structure was determined, SAR and modeling provided insight into the binding interactions. Replacement of the pyridyl side chain by pyrimidine and pyrazine was tolerated with a slight loss in potency, while the use of five-membered N-heterocycles was detrimental to activity. Methylation of the urea NH furnished a completely inactive compound. Constraining and opening the piperidine ring was also tolerated, while increasing the size of this saturated ring system by one methylene group led to a substantial potency loss.
Figure 28
Figure 28. Optimization of covalent-reversible FGFR4 inhibitors possessing an aldehyde warhead.
The advanced lead compound 73d was further optimized to furnish clinical candidate FGF401 (74), a compound with a good oral bioavailability, PK, and safety profile, which is currently in phase I/II clinical studies (NCT02325739) for FGFR4 and β-klotho positive solid tumors and hepatocellular carcinoma.(204) Although the final optimization steps as well as the outcome of clinical evaluation remain yet to be reported, the presented preclinical investigations emphasize that aldehydes, despite potential metabolic liabilities, can indeed be valuable warheads for the covalent-reversible targeting of cysteines beyond the active sites of hydrolases.
2.13. Reversible Cysteine Addition to Activated Nitriles
Besides aldehydes, nitriles have a long history as covalent-reversible warheads for protease inhibitors(196,205) and are employed in approved drugs such as saxagliptin.(206) As indicated by their prevalence in noncovalent inhibitors,(207) nitriles behave relatively inert, and covalent adduct formation generally requires highly reactive active site nucleophiles along with precise positioning of the electrophilic carbon atom. However, the electrophilicity of the nitrile group can be increased by the attachment to electron-withdrawing moieties, e.g., heteroaryl rings, alkylamines,(12,208) or acylated N,N′-dialkylhydrazines.(209)
Pyridine and pyrimidinecarbonitriles show tunable reactivities toward glutathione, which are in a similar range as the ones of acrylamides (Figure 29A,B), suggesting these structural elements as suitable CRGs for targeting noncatalytic cysteines. Nitriles reversibly react with cysteines to form thioimidates via a Pinner-type addition, as it has been shown by X-ray crystallography for the cysteine proteases cathepsin S (PDB 3N3G)(210) and the adenovirus protease adenain (PDB 4PIQ).(211) In the case of 2-pyridinecarbonitrile analogues, a double activation mechanism involving the pyridine nitrogen atom as a general base to deprotonate the cysteine while concurrently activating the nitrile group has been proposed.(210) A recent study aiming to capitalize on the increased reactivity of 2-pyridinecarbonitriles compared to their phenyl counterparts was published by Pamela England and co-workers.(212) They hypothesized that a nitrile group present in the approved nonsteroidal antiandrogen bicalutamide (compound 75, Figure 30A) could be activated to react with the proximal Cys784 in the androgen receptor (AR), a residue that is not among the over 160 point mutations, which have been associated with prostate cancer. DFT calculations predicted a 10-fold increase in electrophilicity by introduction of a pyridine nitrogen atom ortho to the bicalutamide nitrile group (compound 76a). Reactivity studies with 76a and free cysteine in pH 7.4 buffer showed a 99% conversion to the corresponding thiazoline within 4 h, while no S-adduct formation was observed for 75 after 24 h. The additional nitrogen atom increased binding affinity approximately 150-fold (KI = 0.15 nM) compared to the parent compound bicalutamide. In a cellular luciferase reporter assay detecting AR-mediated transcription, a substantial increase in activity (IC50 = 0.015 vs 0.31 μM for 76a and 75) was confirmed, while the two analogues 76b and 76c, devoid of the nitrile group, were much weaker inhibitors (IC50 = 1.41 and 1.33 μM, respectively). A mechanism was proposed in which Arg752 activates the nitrile group via hydrogen bonding (Figure 30B). Although not explicitly mentioned, further activation could result from a hydrogen bond between the pyridine nitrogen atom and the cysteine’s thiol, as mentioned above,(210) to facilitate the nucleophilic attack. NMR studies to confirm the covalent-reversible binding, however, were hampered by protein precipitation, and mutation of Cys784 prevented proper folding. Furthermore, no structural data was reported and an ultimate proof of the suggested covalent interaction remains elusive.
Figure 29
Figure 29. Reactivities of activated nitriles in a GSH-based assay. Half-lives (increasing from left to right) were determined in the presence of 10 mM GSH at pH 7.4 and 37 °C. Data for common acrylamides are provided for comparison.
Figure 30
Figure 30. Bicalutamide-derived antiandrogens with a putative covalent-reversible binding mode. (A) Chemical structures of the compounds studied by England and co-workers. (B) Suggested mechanism of covalent binding to Cys784 of the androgen receptor.
Cyanamides show similar reaction kinetics with GSH as acrylamides (see Figure 29) and should therefore be applicable in nonactive site cysteine targeting. The reversible thiol addition furnishes isothioureas, as it has been confirmed for cathepsin K by X-ray crystallography (PDB 1YK7).(213) While cyanamides are not uncommon in cysteine protease inhibitors,(213−216) reports on targeting noncatalytic cysteines using this functionality are rare. When designing cyanamide-based warheads, it should be kept in mind that free protons at the amino group could, in principle, enable the tautomerization to carbodiimides,(217) which have distinct reactivities. It remains to be tested if this equilibrium is relevant under physiological conditions.
Micah Benson and colleagues from Pfizer reported the cyanamide-based inhibitor PF-303 (77, Figure 31), which was used as a chemical in vivo probe to investigate the phenotype of BTK inhibition in mice.(218) The underlying medicinal chemistry program was not published, but PF-303 was claimed to be highly potent (IC50 = 0.64 nM) and orally bioavailable. The compound, which can be considered as a pseudorigid cyanamide analogue of the covalent BTK inhibitor ibrutinib, possessed a kinact/KI ratio of 1.44 × 105 M–1 s–1, which is in the same range as for many acrylamide-derived kinase inhibitors.(21,219) Binding was reversible with a dissociation half-life of 5 h. The compound also inhibited the kinases BMX and TEC featuring an equivalently positioned cysteine. In contrast, an over 10000-fold selectivity against JAK3 and ITK also sharing this cysteine was observed. The latter finding is in line with SAR reported for a series of ibrutinib-derived ITK inhibitors, where replacement of the acrylamide moiety by cyanamide completely eroded the inhibitors’ activity.(220) PF-303 showed high selectivity against the related Src family kinases and potently inhibited anti-IgM F(ab′)2-promoted B-cell proliferation with an IC50 of 2 nM.
Figure 31
Figure 31. PF-303, a covalent-reversible BTK inhibitor featuring a cyanamide warhead.
2.14. Reversible Cysteine Addition to Isothiocyanates
Isothiocyanates have long been known as natural electrophilic compounds in foods, and their presence has been associated with health benefits. They mainly occur in cruciferous vegetables as metabolic breakdown products of glucosinolates.(221) As a representative of this compound class, allyl isothiocyanate (78, Figure 32A) causes the pungent taste of mustard and wasabi, which has been attributed to TRPA1 channel activation by covalent cysteine (and lysine) binding.(222) Various clinical trials in all phases have been conducted with phenethyl isothiocyanate (PEITC, 79)(221) and sulforaphane (80), e.g., for cancer chemoprevention and the treatment of schizophrenia and autism spectrum disorders (see https://clinicaltrials.gov). Evaluation in a clinical setting and the natural abundance in foods suggest that these electrophiles are well tolerated, however, their biological action has been attributed to multitarget effects. Isothiocyanates rapidly undergo GSH addition to form dithiocarbamates, but this process is reversible(223,224) and trans-thiocarbamoylation to proteins occurs.(225) Reactivity toward amino groups to form stable thioureas is much lower. Nevertheless, lysine residues can be labeled as a consequence of the dithiocarbamate–isothiocyanate equilibrium,(226) but also direct reaction pathways seem possible (Figure 32B). Reactivity of isothiocyanates can be expected to increase with the attachment of electron-withdrawing groups, while electron-donating (+I) substituents should decrease the electrophilicity of the sp-hybridized carbon center.(224) Many proteins, such as glutathione S-transferase π (GSTP1),(227) the cysteine-rich protein Keap1,(228) protein tyrosine phosphatases,(229) or the protein kinase MEKK1,(230) are covalently labeled by isothiocyanates at cysteine residues. Binding to cysteines of tubulin has been suggested as a key mechanism of action of natural isothiocyanates,(231) and the N-terminal catalytic proline in macrophage migration inhibitory factor (MIF) is also modified by these compounds.(232) Because of the reversible nature of cysteine binding, investigation of targets in living systems is challenging. Despite of the wealth of data on isothiocyanates that has accumulated during several decades, systematic studies of SAR and applications in TCI design remain sparse. This might, at least in part, be attributed to the very complex equilibria driving isothiocyanate distribution and availability in vivo.(233)
Figure 32
Figure 32. Isothiocyanates as covalent-reversible warheads for cysteine and irreversible CRGs for lysine. (A) Common isothiocyanates found in cruciferous vegetables. (B) Reversible reaction of isothiocyanates with GSH or cysteines in proteins and slow thiourea formation, e.g., with lysine. Possible direct reaction pathways are depicted as dashed arrows.
2.15. Reversible Cysteine Addition to Electron-Deficient Heteroarenes Forming Stable Meisenheimer Complexes
In a recent publication, Campbell McInnes and co-workers described fragment-like covalent-reversible polo-like kinase (PLK) 1 inhibitors based on a benzothiazole N-oxide scaffold.(234) This compound class was identified by a virtual screening campaign and the initial hit 81a (Figure 33) possessed a moderate potency in the low micromolar range (IC50 = 2.5 μM). Subsequent SAR studies identified the nitro group in the C7-position and N-oxide as crucial features for activity, while replacement of the C2-carboxamide moiety by a nitrile or a hydroxamate group was tolerated with a moderate loss in potency. Lipophilic −I substituents were favorable at the C5 position, with the more potent compound 81b (IC50 = 0.4 μM) featuring a trifluoromethyl thioether group. Because compound 81b possessed limited ATP competitivity and docking suggested close proximity between the Cys67 side chain and the C4-aryl carbon atom, covalent binding via formation of a Meisenheimer complex was considered as the potential mechanism of action. It is worth mentioning that Meisenheimer complex formation has already been described earlier as the mode of action of the glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol.(235) The stability of such negatively charged σ-complexes can be rationalized by the very electron-deficient nature of the (hetero)aryl system combined with the low mobility of the leaving group, preventing its departure. It can further be assumed that a positive electrostatic surface potential in the protein binding pocket would be beneficial to stabilize such complexes. By using n-butylamine and 1-butanethiolate as surrogate nucleophiles in NMR and UV–vis experiments, potency was roughly correlated with the propensity of the compounds to form Meisenheimer-type adducts. As expected, the reaction was reversible upon removal of excessive n-butylamine. Consequently, it can be assumed that such inhibitors would be subjected to rapid but reversible GSH addition in vivo. Moreover, inhibitor 81a was completely inactive (IC50 > 100 μM) against the PLK1 C67S mutant. Screened at 100 μM, 81a showed good selectivity in two small kinases panels, including three kinases with an equivalently positioned cysteine. No details on cellular permeability, metabolic stability, and binding kinetics were provided, and no ligand-bound X-ray crystal structure was solved to confirm covalent modification of Cys67. Although being highly interesting from a conceptual point of view, it remains to be seen to what extend moieties capable of forming stable Meisenheimer complexes, which typically contain one or several nitro groups to stabilize the negative charge, will enrich our current medicinal chemistry toolbox of covalent-reversible warheads.
Figure 33
Figure 33. Meisenheimer complex-forming electrophiles as putatively covalent-reversible PLK1 inhibitors. Mechanism and selected compounds.
2.16. Reversible Cysteine Targeting by Disulfide Tethering
The formation of disulfide bridges is a key feature of cysteine residues in biological systems. Accordingly, cysteines can be addressed by strained or unstrained disulfides via thiol–disulfide exchange, by different reagents possessing electrophilic sulfur atoms, or even with free thiols under oxidizing conditions (see Figure 34A for some examples). Interesting recent applications include disulfide tethering for identifying low affinity fragments,(182,236) thiol-mediated uptake approaches,(237) and glutathione-responsive prodrugs.(238) Disulfide tethering can be very useful in structural biology.(239) For example, covalent trapping of the β2 adrenergic receptor, equipped with an engineered cysteine (H93C), by the disulfide-based ligand FAUC50 (82, Figure 34B) enabled the first X-ray crystallographic structure determination of this GPCR in an agonist-bound state.(240) Although many medicinal chemistry campaigns, especially in academia, have exploited disulfide formation for covalent cysteine targeting (for some recent examples, see refs (241−243)), it remains questionable if this linkage strategy is suitable for targeted covalent inhibition in vivo due to the complexity of physiological thiol–disulfide equilibria and redox systems.(244,245) Therefore, no further discussion is provided.
Figure 34
Figure 34. Reversible cysteine targeting by disulfide bond formation. (A) Selected examples of headgroups known to generate disulfide bonds. (B) FAUC50, a covalent ligand which enabled crystallographic structure determination of the β2 adrenergic receptor.
3. Nucleophilic Targeting of Oxidized Cysteines with Carbon Acids
ARTICLE SECTIONS
Cysteine oxidation is known as an important regulator of protein function.(57) Oxidation of the thiol group with reactive oxygen species (ROS) furnishes sulfenic acids, which can further react following a variety of reversible and irreversible reaction pathways.(246) Reversible S-sulfenylation is involved in the regulation of phosphatases, kinases, ion channels, and a plethora of other proteins.(247) For example, sulfenylation of Cys797 in the EGFR kinase domain increases kinase activity while rendering the target insensitive to modification by covalent EGFR inhibitors.(248) In contrast to thiols, sulfenic acids are only weak nucleophiles while holding a more pronounced electrophilic reactivity. The latter has been capitalized for sulfenic acid detection with nucleophilic carbon acids.(249) The most prominent sulfenic acid sensors are based on 5,5-dimethyl-1,3-cyclohexanedione (dimedone, 83, Figure 35) and similar cyclic 1,3-dicarbonyl scaffolds. Other electrophilic groups that have been shown to trap sulfenic acids include norbornenes(250) and strained cycloalkynes,(251) the latter having simultaneous electrophilic reactivity toward thiols.(247)
Figure 35
Figure 35. Carbon acids adressing cysteine sulfenic acids. (A) Model system used by the Carroll group to assess sulfenic acid labeling with C-nuclophiles. A possible alternative reaction pathway is highlighted by the dashed arrow. (B) Cysteine sulfenic acid formation by ROS and trapping with dimedone. (C) Reactivities of cyclic C-nucleophiles. (D,E) Reactivities of linear C-nucleophiles. (F) Tofacitinib, an approved JAK inhibitor shown to react with sulfenic acids. Reactivity is expressed by pseudo-first-order rate constants derived from an LC-MS assay using the model system shown in (A).
The most notable contributions in targeting oxidized cysteines have come from the group of Kate Carroll, who made extensive use of α-CH acidic β-dicarbonyls and similar compounds for sulfenic acid sensing. For example, they used redox-based probes to monitor oxidation of the phosphatases’ active site cysteine in living cells.(249) As the reaction rate of sulfenic acids with dimedone is relatively low, the Carroll group developed a convenient LC-MS-based assay for screening the reactivity of C-nucleophiles toward a dipeptide-derived sulfenic acid under aqueous conditions. The principle relies on the in situ generation of the sulfenic acid from a more stable cyclic sulfenamide (Figure 35A).(247) A large number of cyclic(247) and linear(252) C-nucleophiles were screened. For cyclic C-nucleophiles, an increase in reactivity could be achieved, for example, by annellating a benzene ring and replacing one of the ketones by a carboxamide or sulfonamide group. The most reactive cyclic C-nucleophiles outperformed the reactivity of dimedone more than 200-fold as indicated by their pseudo-first-order rate constants determined in the above-mentioned assay (see Figure 35C for selected examples). The reaction products were stable against reduction with DTT, GSH, and TCEP, and no cross-reactivity with protected cysteine, cystine, serine, lysine, or a sulfinic acid was observed. However, it should be mentioned at this point that recent evidence suggests that sulfenamides can react faster with dimedone analogues than sulfenic acids.(253) Therefore, the reactivities observed in the aforementioned assay may at least in part arise from direct reaction with the employed sulfenamide precursor. In a model study, selected compounds showed near-quantitative labeling of the C64S/C82S mutant of the glutathione peroxidase (GPX) 3 at a single oxidized cysteine residue. Linear C-nucleophiles could also be tuned, however, within a narrower range (Figure 35D,E). Good reactivities were obtained by attaching electron-withdrawing α-groups to methyl sulfones. Interestingly, the reaction products of the most reactive derivative 85 could be readily cleaved under reducing conditions, a finding that was confirmed in a GPX3-SOH model and in HeLa cell lysates. In contrast, nitro analogue 84 formed stable products withstanding reductive cleavage for several days. Intriguingly, the cyanoacetamide group of the approved reversible JAK inhibitor tofacitinib (86, Figure 35F) was also shown to react with cysteine sulfenic acids, raising the question about potential off-target modification.
In a subsequent study, the same group investigated the cysteine “sulfenylome” of RKO colon adenocarcinoma cells.(254) To this end, five of the C-nucleophiles from the above studies were equipped with alkyne handles (87–91, Figure 36) to enable click-chemistry-based tagging and enrichment. By employing MS-proteomics, 1283 S-oxidation sites in 761 proteins were identified. Because sulfenamides, which can be formed from sulfenic acids, but also from (mixed) disulfides and amides, are quite abundant in biological systems, it seems likely that the observed oxidation sites account for a combination of the two former oxidized species.(253) Nevertheless, little overlapping was observed with respect to the labeling sites of different nucleophiles, suggesting significant discrimination even at this low level of structural complexity. Despite the relatively slow reaction kinetics, these data indicate that selective targeting of cysteine sulfenic acids and sulfenamides with more optimized compounds should in principle be possible. Further investigations, including the application of such molecules in vivo, are highly anticipated.
Figure 36
4. Targeting the Lysine Side Chain
ARTICLE SECTIONS
Significant effort has recently been put into developing selective warheads for lysine residues, and key studies up to early 2017 have been summarized in a recent mini-review.(39) Lysine, together with cysteine, has been the most common residue addressed in bioconjugation chemistry.(41) Perhaps the most prominent labeling reagents for the lysine ε-amino group are highly activated N-hydroxysuccinimide (NHS)-esters, which are generally considered too reactive for application in TCIs. Lysine offers certain distinct advantages over cysteine, most notably the much higher abundance (5.8% vs 1.9%).(59) It is typically found on protein surfaces, on interfaces mediating protein–protein interactions as well as in binding cavities. Lysine residues play a role in catalysis, e.g., by acting as a base or as a nucleophile.(255) Moreover, lysines in active sites can assist catalysis by positioning reactive residues or activating them via (charge-assisted) hydrogen bonds (e.g., in kinases). In such cases, lysine residues are indispensable, and resistance mutations, as they may occur for noncatalytic cysteines,(65) unlikely. However, lysine has been much less frequently considered as a residue to be addressed by TCIs. The latter fact can be attributed to the special challenges associated with the ε-amino group as a nucleophile, especially when compared to cysteine. At physiological pH, surface-exposed lysine (pKa ≈ 10.5)(256,257) is almost entirely protonated (99.9%), rendering the side chain poorly nucleophilic. However, the pKa largely depends on the chemical environment and buried lysines can undergo a pKa shift of up to 5 units, making them addressable by electrophiles.(257) As discussed before (see section 2.4 and section 2.13), the ligand itself may also contribute to pKa perturbation. Besides protonation, post-translational modifications (most notably acylation) can make the lysine side chain unsusceptible toward electrophilic reagents. Another special feature of lysine is the long and linear architecture of the side chain causing a high degree of conformational freedom. Whether this property is to be considered favorable or not cannot be generalized and depends on the respective context.
By employing a similar approach as previously used for assessing the reactivity of cysteines in native biological systems,(13,156) the Cravatt group set out to identify ligandable lysine residues by means of chemical proteomics.(258) A highly activated ester probe equipped with an alkyne handle (compound 92, Figure 37) was used to quantify the reactivity of lysine residues in proteomes. The study recovered over 9000 reactive lysines from different human cell lines, a much higher number than previously found for cysteine with a iodoacetamide probe, that roughly reflects the relative abundance of these amino acids.(13,156) Experiments at different probe concentrations and competition experiments with various reactive fragments (general structures 93–95, Figure 37) identified (hyper)reactive lysine residues that have a high probability of being addressable by small molecules in a selective manner. A fact particularly worthy of note is that such lysines were identified mainly in proteins not found in the DrugBank, including many challenging targets such as transcription factors and scaffolding proteins. However, no conserved motifs could be associated with lysine hyper-reactivity, and further insights are required to deepen our understanding of lysine nucleophilicity and ligandability.
Figure 37
Figure 37. Probes and warheads used to indentify ligandable lysines by chemical proteomics.
Lysine side chains can react covalently with Michael acceptors, but aza-Michael additions with lysines have often been discovered by chance rather than implemented by design. For example, the natural product wortmannin (96, Figure 38) labels Lys833 in phosphoinositide 3-kinase γ (PI3Kγ) by aza-Michael addition and concomitant opening of the ligand’s furan ring (Figure 38 and PDB 1E7U).(259,260) More recently, the Cheeseman group serendipitously discovered that an adenosine-derived inhibitor designed to target a cysteine (Cys17) residue in the heat shock 70 kDa protein 1 (HSP72) was in fact bound to a lysine side chain (Lys56).(261) In general, most Michael acceptors are more reactive toward the “softer” cysteine thiols (vide infra), which can be problematic as targeting the lysine ε-amino group may require more activated acceptors. The latter are more likely to exhibit cysteine-mediated off-target reactivity. The aza-Michael reaction with lysine furnishes Mannich-type bases which can re-eliminate the (protonated) amino group to recover the reactive electrophile. Thus, adduct formation can in principle be considered reversible.(262) The extent of the reverse reaction, however, largely depends on the chemical nature of the reaction product (i.e., α-CH acidity of β-amino ketones vs esters or amides and the basicity of the amino group) and it remains to be investigated if it could become relevant under physiological conditions.
Figure 38
Figure 38. Reaction mechanism of wortmannin with Lys833 in PI3Kγ.
4.1. Reactivities of Michael Acceptors and Nitriles toward Lysine versus Cysteine
Adam Gilbert and colleagues from Pfizer have recently assessed the reactivities of common electrophiles including α,β-unsaturated amides, α,β-unsaturated sulfones, α,β-unsaturated sulfonamides, cyanamides, and nitriles toward N-α-acetyl lysine and compared these with the corresponding GSH reactivities.(263) In the case of N-α-acetyl lysine, the assay was conducted at pH 10.2 (≈ 20% of unprotonated amine) to mimic the pKa perturbation in binding pockets while reactions with GSH were performed at pH 7.4. Although it is clear that this model system does not fully reflect the real situation in binding sites, it provides a reasonable picture on reactivity trends toward thiol(ate) and amino nucleophiles. Remarkably, no reaction was observed for N-α-acetyl lysine at pH 7.4 even with the most reactive electrophiles, pointing out the poorly nucleophilic nature of the protonated amine and confirming that the carboxylate group also remains inert. As expected, this model system recapitulated the same general trends as previous GSH reactivity studies (for selected examples, see Figure 39).(263) Reactions of α,β-unsaturated amides proceeded 1.2–5 times faster with glutathione than with N-α-acetyl lysine. Similarly, the GSH addition to N-cyanopyrrolidine was more than twice as fast compared to N-α-acetyl lysine, while the reaction of GSH with 2-cyanopyrimidine was even nine times faster. Interestingly, an opposite tendency was observed for α,β-unsaturated sulfones and the corresponding sulfonamides. For example, α,β-unsaturated sulfonamides reacted 1.6–4-fold faster with N-α-acetyl lysine compared to GSH, while an even nine times faster reaction was measured for methyl E-prop-2-enyl sulfone. This difference may be rationalized with the Pearson HSAB theory,(54) suggesting that the strongly electron-withdrawing sulfonyl group makes the β-position less polarizable (i.e., harder), favoring the reaction with a harder nitrogen nucleophile rather than with the soft thiol(ate) group. Although α,β-unsaturated sulfones and sulfonamides have a higher general reactivity compared to the corresponding acrylamide-derived Michael acceptors, sterical and electronic tuning of these electrophiles might be harnessed to generate selective lysine-targeted probes with a low degree of promiscuity.
Figure 39
Figure 39. Reactivity of different Michael acceptors and activated nitriles toward lysine and GSH. Compounds with a preference for GSH are shown the upper row, while such preferably reacting with N-α-acetyl lysine are depicted in the lower row. Half-lives were determined in the presence of 50 mM N-α-acetyl lysine at pH 10.2 and 37 °C or 10 mM GSH at pH 7.4 and 37 °C.
The higher reactivity of α,β-unsaturated sulfones toward lysine was used by the groups of Bernard Golding and Jane Endicott to generate the first irreversible inhibitor of cyclin-dependent kinase (CDK) 2.(264) The key compound of this study, vinyl sulfone NU6300 (98a, Figure 40A), was identified while optimizing sulfonamide NU6102 (97). Binding of 98a was reversible on short time scales, and an equilibrium binding constant could be determined by SPR (KD = 1.31 μM), pointing out the only moderate affinity. However, inhibition was time-dependent, and 20 h of incubation with 98a abolished 50% of the apparent binding capacity of the slightly more potent saturated analogue NU6310 (98b, KD = 0.72 μM). Mass spectrometry and washout experiments confirmed the covalent modification. The binding to nonconserved Lys89 was suggested by studies with the CDK2 K89V mutant, which did not show adduct formation in MS experiments and recovered activity upon removal of the compound by dialysis. X-ray crystallography finally confirmed the binding mode (Figure 40B). The compound inhibited 13 out of 113 kinases in a panel at 1 μM but only two of those in a time-dependent manner. The rather slow inactivation kinetics might be rationalized by the surface-exposed location of Lys89, suggesting that the ε-amino group is almost completely protonated and therefore unreactive. However, it remains questionable if such slow inactivators would be useful in an in vivo setting as they only partially benefit from key advantages of covalent labeling (e.g., decoupling of pharmacodynamics from pharmacokinetics) while comparatively rapid off-target modification might be an issue, especially when quite reactive electrophiles are employed.
Figure 40
Figure 40. Vinyl sulfone targeting a lysine in the solvent-exposed front region of CDK2. (A) Covalent and noncovalent inhibitors. (B) X-ray crystal structure of vinyl sulfone 98a covalently bound to Lys89 flanking the solvent-exposed front region of CDK2 (PDB 5CYI). The purine NH is hydrogen-bonded to the backbone carbonyl atom of Glu81, while the N3-atom and the diaryl NH are anchored to the backbone of Leu83 by two additional hydrogen bonds. Another water-bridged hydrogen bond links the purine N7-atom to the backbone NH group of Asp145 in the DFG motif. Further direct and water-mediated hydrogen bonds are established by the sulfonyl group. The N-terminal lobe was omitted for clarity.
4.2. Reactivities of Sulfonyl Fluorides and Related Sulfur (VI) Fluorides toward Lysine, Tyrosine, and Cysteine
Sulfonyl fluorides are among the most prominent electrophiles for addressing the ε-amino group of lysines. They have also been used to target tyrosine, activated serine, or threonine residues and are reactive toward cysteine and histidine side chains as well.(265) The potential of sulfonyl fluorides, which have been known as insecticides for about 100 years,(266) in drug discovery was first recognized by Bernard Baker in the late 1960s.(267) However, until its recent revival by Sharpless and co-workers, sulfur (VI) fluoride chemistry (sulfonyl fluorides/chlorides are interpreted as sulfur (VI) halides in this context) has played a subordinate role while sulfonyl chlorides have frequently been used as reagents in organic synthesis.(266) The high reactivity of sulfonyl chlorides, as well as their instability toward hydrolysis and reduction, have hampered their use for biological applications. In contrast, sulfur (VI) fluorides are thermodynamically stable and less prone to hydrolysis and reduction. Unlike their chloride analogs, they are quite demanding electrophiles requiring sufficiently strong nucleophiles and proper solvation of the fluoride group for reaction. While sulfonyl chlorides may also be attacked on the chlorine atom, sulfonyl fluorides exclusively react at the sulfur atom. Therefore, sulfur (VI) fluoride exchange (SuFEx) has recently manifested itself as a useful click-type chemical reaction(266) with favorable properties for labeling biomolecules.(45) Sulfonimidoyl fluorides, the imino-analogues of sulfonyl fluorides, possess similar favorable properties and an additional handle to attach substituents, allowing for modulation of reactivity by the moiety attached to the nitrogen atom. Fluorosulfates and the analogous sulfamoyl fluorides are even less reactive (Figure 41). It has been further suggested that fluorosulfates only become activated if the departing fluoride ion is stabilized by the surrounding protein, e.g., by a hydrogen bond donor or suitable electric field effects.(268) Therefore, this functional group has been termed a “latent electrophile.”(269)
Figure 41
Figure 41. Sulfur (VI) fluorides for lysine and tyrosine targeting.
Early examples of the application of sulfonyl fluorides in chemical biology and drug discovery include protease labeling reagents such as benzenesulfonyl fluorides (99a,b, Figure 42)(270,271) and dansyl fluoride (100),(272) enzyme inhibitors exemplified by dihydrofolate reductase inhibitors (e.g., NSC 127755, 101) and nucleotide-derived probes (e.g., 5′-p-fluorosulfonylbenzoyl adenosine, 5′-FSBA, 102).(273) Sulfonyl fluorides have also been used to generate irreversible GPCR ligands,(274−276) and the tyrosine-targeted covalent adenosine A1 receptor antagonist DU172 (103) has recently enabled the crystal structure determination of the latter receptor.(277) Methanesulfonyl fluoride (MSF, 104) is known as an irreversible acetylcholinesterase (AChE) inhibitor for more than 50 years.(278) On the basis of the rationale that AChE in the CNS has a substantially lower resynthesis rate than in peripheral tissue, clinical phase I and II studies have been conducted assessing the potential of MSF in the treatment of Alzheimer’s disease.(279,280) MSF doses up to 0.18 mg/kg three times a week were well tolerated while effectuating the desired AChE inhibition in vivo and improving cognitive performance. Further applications of sulfonyl fluorides have been reviewed recently.(39,265,266,281)
Figure 42
Figure 42. Examples of sulfonyl fluorides applied in medicinal chemistry and chemical biology.
A detailed assessment of the reactivity of sulfonyl fluorides and analogous sulfur (VI) fluorides (Figure 43) was provided by Neil Grimster and colleagues from AstraZeneca.(282) They found that arylsulfonyl fluorides readily react with N-acetylcysteine (NAC) at pH 7.5. Reaction rates of most derivatives were slightly higher compared to analogous N-aryl acrylamides. However, the reactivity of sulfonyl fluorides could be tuned over a wider range with a variety of electron-withdrawing and electron-releasing groups (Figure 43A), the rate following a log–linear relationship to the Hammett parameter of the aryl substituent. The higher sensitivity to the electronic properties of the aromatic ring system compared to α,β-unsaturated amides can be rationalized by the direct attachment of the sulfonyl fluoride group. The substitution products, however, were unstable under the assay conditions, suggesting this functionality to be unsuitable for durable cysteine modification. This finding is not unexpected because the thiosulfonate S-esters formed by fluoride displacement are known to be unstable toward hydrolysis and react with thiols to form disulfide bonds (see the mechanism in Figure 43D).(283) In cells, an analogous reaction would predominantly produce glutathione disulfide (GSSG) due to the high intracellular GSH concentrations. It can also be assumed that thiosulfonate S-esters within protein binding sites would be cleaved by reductive sample preparation, emphasizing that cysteine labeling in living systems would be difficult to capture. The reactivity of sulfonyl fluorides toward N-acetyl tyrosine and N-α-acetyl lysine was considerably lower than toward NAC, but stable substitution products were obtained in both cases. While the initial reaction rate of phenylsulfonyl fluoride (105a) with N-acetyl tyrosine was 3.4 times decreased compared to NAC, a 10-fold slower reaction was observed for N-α-acetyl lysine at pH 7.5. Reaction rates increased substantially when the pH was increased, reaching a similar level for tyrosine and lysine at pH 10 (cysteine reacted too fast for rate determination at this pH). However, no reaction other than hydrolysis was observed for N-acetylated serine. Warhead hydrolysis (pH 7.5) was rapid for electron-deficient arylsulfonyl fluoride derivatives (t1/2 ≈ 5–15 min), while analogues bearing electron-donating substituents were stable up to several days. Nevertheless, hydrolysis was much slower than reaction with the tyrosine side chain. As shown for N-acetyl tyrosine, the vinyl sulfonyl fluoride 106 was 9 times less reactive than 105a with no concomitant 1,4-addition being observed. The benzylic derivative phenylmethylsulfonyl fluoride (PMSF, 107), a reagent commonly used in biochemistry to label active site serine residues or to prevent protein degradation in cell lysates,(284) was rapidly hydrolyzed, presumably due to its α-CH acidity favoring the reaction via a sulfene-like intermediate. Plasma stabilities reflected the trend from the hydrolysis assay with 4-dimethylamino derivative 105d showing a favorable half-life (t1/2 = 25.1 h). Clearance data was obtained for compounds 105b–d (165, 24 and 170 × 10−6 μL/min, respectively) and several 5′-FSBA analogues (vide infra) in rat hepatocytes. In the latter series, metabolic clearance correlated rather with the lipophilicity of the compounds than with the Hammett parameters of the aryl substituents, suggesting that inherent warhead reactivity is not the key determinant of hepatic metabolism.
Figure 43
Figure 43. Compounds used for assessing the reactivity of sulfur (VI) fluorides toward different amino acids. (A) sulfonyl fluorides. (B) sulfonimidoyl fluorides. (C) aryl fluorosulfates. (D) Mechanism of the reaction between phenylsulfonyl fluoride and NAC.
A series of related sulfonimidoyl fluorides (represented by 108a–f, Figure 43B) with different substituents at the nitrogen atom was also probed for reactivity toward N-acetyl tyrosine. As expected, these compounds featured a lower reactivity compared to the analogous sulfonyl fluorides and electron withdrawing N-substituents were required for obtaining substitution products at the given conditions. Among the activated derivatives, N-acylated derivatives 108a,b reacted slightly faster than sulfonyl fluoride 105a, while the N-Boc analogue 108c reacted at a similar rate. N-Methylated analogue 108f showed no reaction under the same conditions. Interestingly, hydrolytic stability was increased compared to 105a even for the more activated derivatives, suggesting this structural class as superior warheads in terms of stability, versatility, and tunability. However, it should not remain unmentioned that electron-rich N-aryl derivatives (e.g., compound 108d), in contrast to p-CF3-phenyl-substituted analogue 108e, degraded rapidly, presumably via an oxidative decomposition pathway. NAC also reacted with the N-acylated derivatives to form unstable products while N-alkylated derivatives remained inert. Aryl fluorosulfates 109 and 110 (Figure 43C) were completely stable at pH 7.5, and neither hydrolysis nor substitution with N-α-acetyl lysine or tyrosine occurred within a 24 h time frame. Reaction with NAC was very slow, but extended incubation (4 days) resulted in the formation of the corresponding phenol, presumably via cystine formation in analogy to Figure 43D and subsequent loss of SO2. It is worth mentioning that the intrinsic reactivity of fluorosulfates is so low that fluorosulfate-l-tyrosine could recently be genetically encoded and incorporated into proteins as a latent electrophile for inter- and intraprotein cross-linking in mammalian cells.(285) No sulfamoyl fluorides were evaluated in this study, but it can be assumed that these compounds would even be less reactive than the corresponding aryl fluorosulfates.(266)
4.3. Lysine Targeting with Sulfur (VI) Fluorides
In the second part of the last-mentioned study, the reactivity of sulfonyl fluorides in a protein binding pocket was probed. To this end, analogues of the above-mentioned inhibitor 5′-FSBA (m-5′-FSBA; general structure 111, Figure 44) were prepared with different substituents ortho to the warhead. A crystal structure of prototype inhibitor 111a in complex with FGFR1 (PDB 5O49) and MS experiments confirmed the expected binding to the conserved Lys514 and suggested that an ortho-substituent could be accommodated by the binding site. The rate of covalent modification was evaluated by an LC-MS method showing that steric bulk in the ortho position did not significantly influence the reaction rates, which roughly correlated with the substituent’s Hammett parameters. As expected, two analogous aryl fluorosulfate derivatives showed a very low reactivity with only traces of covalent modification being detectable after extended reaction times.
Figure 44
Figure 44. Lysine-targeted m-5′-FSBA analogues for the evaluation of the relationship between warhead reactivity and FGFR1 inhibitory activity.
An interesting application of sulfonyl fluorides as CRGs was provided in 2017 by Jack Taunton and co-workers, who generated a lysine-targeted broad spectrum kinase inhibitor as an active-site probe for chemical proteomics applications.(286) A promiscuous pyrimidine 3-aminopyrazole scaffold was selected based on X-ray crystal structures of such ligands in the ATP pockets of various kinases. Structural data indicated that a suitable linker attached to the 2-position of the pyrimidine ring would position a phenylsulfonyl fluoride proximal to the conserved active site lysine, a residue that is involved in the positioning and activation of the ATP triphosphate moiety. Using SRC as a model kinase, it was shown by MS and X-ray crystallography that optimized and clickable probe XO44 (112, Figure 45A) specifically modified the expected lysine (Lys295, Figure 45B), while none of the other 16 lysines and 13 tyrosines were labeled by a 3-fold excess of the compound. Another X-ray crystal structure (PDB 5U8L) confirmed a similar binding mode for the EGFR receptor tyrosine kinase. In Jurkat T-cells, the inhibitor captured 133 protein kinases (of which 50 are not covered by the kinobead technology(287)) and even 219 kinases were inhibited >50% at 1 μM in a panel containing 375 kinases. The increased number of kinases captured in the panel could reflect the expression pattern of kinases in Jurkat cells. On the other hand, it might be an artifact from the nonphysiological conditions of in vitro screening panels or arise from noncovalent inhibition that cannot be captured in the cellular labeling experiment. Although some nonkinase off-targets were also modified, kinases accounted for the bulk of signal in MS experiments. Competition experiments with dasatinib subsequently validated the use of probe 112 for cellular selectivity profiling.
Figure 45
Figure 45. (A) Promiscuous kinase probe XO44. (B) X-ray crystal structure of XO44 covalently bound to the conserved Lys295 in the kinase SRC (PDB 5K9I). The 3-aminopyrazole is anchored to the hinge region by three hydrogen bonds involving the backbone of Glu339 and Met341. The sulfonyl group forms two additional hydrogen bonds with Phe278 and Gly279 in the glycine-rich loop, while the propargyl amide tag is oriented toward the bulk solvent without being involved in specific interactions.
As an application example of aryl fluorosulfates, these warheads were used by Jeffery Kelly and co-workers in fluorogenic probes to image transthyretin in living cells and Caenorhabditis elegans.(288) Cell permeable inhibitors 113a,b (Figure 46A) were designed to target the pKa-perturbed Lys15 side chain, which had previously been addressed with the analogous sulfonyl fluoride 114.(289) Although the probes were well-suited for the envisaged imaging purposes, covalent modification was very slow and only 6% and 29% labeling were observed after 24 h with 113a and 113b, respectively. These findings comply with the known low reactivity of the fluorosulfate group, however, the slow reaction could also be indicative of a suboptimal alignment between the reactants. Despite the slow reaction, MS-based analysis confirmed Lys15 as the site of modification while little general reactivity toward the proteome could be detected. Unexpectedly, the ε-amino group was present as a free sulfamate without the ligand attached (Figure 46B) indicating that the substitution product had been hydrolyzed. Although the authors stress the probable catalytic effect of the target protein on hydrolysis, these results demonstrate the limited stability of such covalent linkages that will have to be evaluated on a case-by-case basis. In proteins where the lysine residue is required for catalytic activity, the ε-sulfamate would still be detrimental to activity while one equivalent of the unreactive ligand with a free phenolic hydroxy group would be released. Further data on the general stability of protein-linked disubstituted sulfates or sulfamates under physiological conditions would be appreciated. Examples for the use of fluorosulfates in tyrosine and serine targeting can be found in the sections 5.1 and 6.1.
Figure 46
Figure 46. Fluorosulfates for lysine targeting. (A) Aryl fluorosulfates and sulfonyl fluorides designed for adressing Lys15 in human transthyretin. (B) X-ray crystal structure of 113b bound to human transthyretin (PDB 4YDM). Unexpectedly, the free Lys15 ε-sulfamate was observed instead of the covalently bound ligand. The ligand is located in a relatively shallow pocket on the protein surface forming only a single conserved hydrogen bond between the hydroxy group of the dichlorophenol moiety and the side chain of Ser117. An alternative orientation of the 3-hydroxyphenyl residue was omitted for clarity.
4.4. Lysine Acylation by Activated Esters
The potential of activated esters for targeting lysine side chains was recently emphasized by a team around Sebastien Campos at GSK.(290) With the aim of generating a potent and selective covalent PI3Kδ inhibitor, they modified the clinical candidate GSK2292767 (115, Figure 47), a highly optimized and isoform-selective reversible PI3Kδ inhibitor. The sulfonamide moiety in 115 had been shown to interact with the conserved Lys779 ε-amino group, suggesting that replacement by an activated ester (116a–f) would be a promising strategy to address this residue. A variety of phenolic esters with electron-withdrawing or releasing substituents in the para position was prepared and tested for their ability to covalently inactivate PI3Kδ. Isoform selectivity and activity in human whole blood were assessed in parallel. Activity in the isolated kinase assay correlated roughly with the electronic properties of the substituents. The 4-nitro derivative 116b proved to be most potent (pIC50 = 9.2) but featured a slightly decreased isoform selectivity and limited stability in DMSO. In contrast, slightly less reactive esters 116a,c,d maintained low nanomolar potency, high isoform selectivity (ca. 2–3 orders of magnitude), and excellent activity (≈ 10 nM) in human whole blood. Kinetic analysis revealed that the rate of covalent inactivation was similar for all compounds (kinact = 5.5–7.5 × 10–3 s–1) and did not correlate with the leaving group properties of the phenolate, while KI spanned a range from 40 nM (compound 116b) to 7.8 μM (compound 116f). Mass spectrometry and X-ray crystallography confirmed the exclusive labeling of Lys779 by 4-fluorophenyl ester 116d, and as expected, the nonactivated methyl ester 116g bound only reversibly to the enzyme. Compound 116d was stable against hydrolysis and reaction with N-α-Boc-lysine at pH 7.4. Interestingly, 116d showed a selectivity window in which PI3Kδ was covalently inhibited while PI3Kα and β engagement was reversible, indicating that covalent bond formation is critically dependent on reversible interactions. The compound showed high selectivity in a panel of 140 protein and 10 lipid kinases and the analogous azide-labeled probe 117 revealed a relatively clean chemoproteomic profile in Ramos cells. Washout experiments in CD4+ T-cells showed sustained suppression of IFNγ release for 48 h upon stimulation with αCD3, indicating prolonged cellular target engagement. However, no data on the metabolic stability of these compounds were reported. It is not unlikely that such activated esters are rapidly inactivated by esterases, which may erode activity in vivo or, following the rationale of Cravatt’s fumarates (section 2.1), improve kinetic selectivity. Finally, it should be noted that activated carbamates that have been frequently employed to target catalytic serines in drug discovery and crop protection(291) might be used in a similar manner as these activated esters.
Figure 47
Figure 47. Development of activated esters covalently targeting Lys779 in PI3Kδ.
4.5. Acylation of Surface-Exposed Lysines by N-Acyl-N-alkyl Sulfonamides
Another structure class that has recently been used to target lysines are N-acyl-N-alkyl sulfonamides. In early 2018, Itaru Hamachi and co-workers reported on the optimization of these moieties to achieve rapid ligand-directed labeling of surface-exposed lysine residues.(292) Activation of the acyl functionality was achieved by attaching electron-withdrawing groups to the sulfonamide nitrogen atom via a methylene spacer (Figure 48A). In an initial experiment, FKBP12 was used as a model protein. SLF, a known FKBP12 ligand (reported KD = 20 nM), was attached via a spacer to the sulfonamide sulfur atom while biotin was employed as the transferable N-acyl moiety and a cyanomethyl residue was utilized as the activating group (compound 118, KI= 210 nM). Incubation of this probe with FKBP12 predominantly biotinylated a single surface-exposed lysine, Lys44, located near the ligand binding site. Reasonable rates (kinact/KI = 2.9 × 104 M–1 s–1, kinact = 6.1 × 10–3 s–1) were achieved with this compound, while the replacement of the nitrile group by 4-nitrophenyl or 2,4-dinitrophenyl slowed down reaction kinetics substantially. Hydrolysis of compound 118 in aqueous buffer at pH 7.2 was slow, with a half-life of 43 h. The probe selectively labeled FKBP12 with biotin in HeLa cell lysates when the probe and the protein were present at an equimolar concentration (both 1 μM). However, increasing the probe concentration or competition with the high-affinity ligand rapamycin suppressed FKBP12 biotinylation and caused unspecific labeling. This underlines again the importance of potent and selective reversible binding for target specificity, especially when highly reactive electrophiles are employed. Remarkably, similar experiments were conducted with an analogous trimethoprim-derived probe (not shown) specifically labeling Lys32 in E. coli dihydrofolate reductase (kinact/KI = 9.3 × 103 M–1s–1, kinact = 1.3 × 10–2 s–1). In an inverse approach, an irreversible Hsp90 inhibitor (120, Figure 48B) was generated by using the known ligand PU-H71 (119) as the transferable N-acyl substituent. A spacer was required to bridge the distance between the target lysine and the ligand binding site. Compound 120 featured a KI of 62 nM and modified Lys58 (kinact/KI = 2.9 × 104 M–1 s–1) as determined by MS. The compound showed durable engagement of Hsp90 in SKBR3 cells, as demonstrated by washout and competition experiments. Encouragingly, an inverted probe featuring a transferable fluorescein marker labeled only four additional proteins, all of those to a lesser extent than Hsp90, indicating good cellular selectivity. Although labeling of surface-exposed lysines, which are typically poor nucleophiles, is a special feature of this compound class, this behavior could not be fully rationalized. Despite the reasonable reaction kinetics and the potential of further tuning by suitable N-alkyl substituents, the CRG is relatively bulky and might be usable only for special applications. For targeting surface-exposed lysine side chains, this bulkiness might not be a major issue, in sharp contrast to such lysines located in spatially restricted binding sites. Finally, despite the good cellular selectivity demonstrated for compound 120, the intrinsic reactivity of this warhead class toward other nucleophiles remains yet to be assessed.
Figure 48
Figure 48. N-Acyl-N-alkyl sulfonamides addressing surface-exposed lysine side chains. (A) Biotin-transferring probes. (B) Covalent ligand design and application example. The transferable residue is highlighted in red.
Further examples of electrophiles that have been used to irreversibly target lysine residues include natural product-derived spiro-epoxides addressing Lys100 in phosphoglycerate mutase 1 (PGAM1)(293,294) and quinazolin-4(3H)-one hydroxamate esters modifying nonactive site lysines in the bacterial serine endoprotease DegS.(295) Chemoselective methods for irreversible lysine modification, such as capture with diazonium terephthalates(296) or ortho-phthalaldehyde-amine condensations,(297) have recently emerged, but these can primarily be considered useful for bioconjugation applications rather than for specific covalent targeting in complex biological systems. The interested reader is referred to recent reviews.(41−43,46)
4.6. Condensation of Lysine with Aldehydes Forming Stabilized Schiff Bases
Schiff bases (imines), which are formed by the reversible condensation of an amine with an aldehyde or ketone, are quite common in biological systems. Prominent examples include pyridoxal phosphate (PLP)-dependent enzymes or the light sensitive GPCR rhodopsin, both binding their cofactors via Schiff base formation.(298) In medicinal chemistry, however, ketones and especially aldehydes are relatively unpopular. One of the main reasons for their avoidance is metabolic liability, as these chemical functionalities are prone to reduction by aldo-keto reductases and short-chain dehydrogenases/reductases or, in the case of aldehydes, oxidation to the corresponding acids by aldehyde dehydrogenases.(299) However, as highlighted in section 2.12, aldehydes can also be surprisingly stable. A recent study showed that metabolically labile aldehydes can be replaced by ketones whose metabolic stability, solubility and, to a lesser extent, the ketone–imine equilibrium can be tuned by the attachment of suitable residues.(199) This strategy furnished novel inhibitors of the cystine–glutamate antiporter, but covalent target engagement was not proven experimentally. Although Schiff base formation is highly reversible, mass-spectrometric analysis of the covalent complex is possible after reducing the Schiff base with borohydride to obtain a stable secondary amine. For example, this technique has been used to demonstrate the covalent binding of the umbelliferon-derived ligand 4μ8C with two distinct lysine residues (Lys509 and Lys907) in the inositol-requiring enzyme (IRE) 1.(200)
The stabilization of Schiff bases by coordination of the nitrogen lone pair to a boronic acid has been pursued by Pedro Gois and co-workers as a strategy for reversible protein modification.(300) In drug discovery, boronic acids are well-known as warheads for catalytic serine or threonine residues, as exemplified by the approved proteasome inhibitor bortezomib.(301) In this study, however, a boronic acid was introduced to the ortho-position of a benzaldehyde moiety in order to obtain an iminoboronate upon Schiff base formation. The latter is stabilized by an intramolecular dative bond between the nucleophilic nitrogen lone pair and the electrophilic boron center (Figure 49). In contrast to many of the above-mentioned methods, iminoboronate formation did not require pKa perturbation and proceeded readily between the model substrate 2-formylbenzeneboronic acid (121) and n-butyl amine at pH 6–9. The reaction was reversed upon the addition of GSH, fructose, and dopamine, suggesting reversibility under physiological conditions, which was later confirmed by Gao and co-workers.(302,303) Proteins such as cytochrome c, ribonuclease a, and myoglobin fully converted with excess 121 within 5 min.
Figure 49
Figure 49. 2-Formylbenzenboronic acid reversibly forming stabilized Schiff bases with amines.
Applications of this chemistry include the labeling of Gram-positive bacteria by targeting amine-presenting lipids(302) and more recently the inhibition of the induced myeloid leukemia cell differentiation protein (MCL-1), a protein–protein interaction target which is a key survival factor for various human cancers.(304) In the latter study, a group of researchers around Qibin Su and Neil Grimster attached a 2-carbonylphenylboronic acid warhead to a known indole-acid based MCL-1 inhibitor to reversibly target the surface-exposed Lys234 side chain. Formylphenylboronic acids 122a,b (Figure 50) as well as the analogous acetophenone 122c featured low nanomolar IC50 values in a TR-FRET assay, while derivatives lacking either the boronic acid or the ortho-carbonyl group were substantially less active. The improved potency translated into superior cellular activity, and the best compound (122b) showed an EC50 of 75 nM in the MCL-1-dependent cell line MOLP-8. Experiments with other MCL-1-dependent cell lines confirmed cellular activity, while MCL-1 independent cell lines remained unaffected. Mass spectrometry confirmed ca. 50% covalent labeling with 122a after 1 h, while acetophenone 122c reacted slightly slower. However, 50% conversion was not exceeded with an excess of 122a even after prolonged exposure, suggesting that an equilibrium stage had been reached. Reversibility was shown by SPR experiments and ligand dissociation fitted a two-state binding model (kd1 = 0.14 s–1 and 0.13 s–1; kd2 = 0.018 s–1 and 0.010 s–1 for 122a and 122b, respectively). It is worth mentioning that the warhead alone did not generate covalent complexes. Although no X-ray data was included, the modification of Lys234 was supported by binding data of covalent and noncovalent analogues to the K234A mutant. As demonstrated by this study, 2-carbonylphenylboronic acids can be used as cell-penetrable warheads that enable the reversible engagement of surface-exposed lysine side chains in the proximity of small molecule binding sites. It will be interesting to see whether addressing buried lysines, e.g., in kinases, will substantially decrease off-rates to enable complete labeling. Finally, the stability of this structure class under physiological conditions, especially with respect to the mentioned susceptibility of aldehydes and ketones toward redox biotransformation, will have to be assessed.
Figure 50
Figure 50. 2-Formylbenzenboronic acids and analogous acetophenones targeting MCL-1 via Schiff base formation.
5. Targeting the Tyrosine Side Chain
ARTICLE SECTIONS
Neutral tyrosine has a relatively low intrinsic nucleophilicity when compared to cysteine or unprotonated lysine. Therefore, the selective modification of nonactivated tyrosine can be challenging. However, its phenolic hydroxy group possesses a slightly higher acidity compared to the protonated lysine side chain (pKa ≈ 9.7 vs 10.5),(305) and tyrosine residues in proteins can have a significantly lowered pKa, favoring the formation of the highly nucleophilic phenoxy anion.(306) Increased reactivity of tyrosine toward sulfonyl fluorides, for example, correlated with the proximity of a basic amino acid residue.(307) The phenoxy anion is a relatively hard nucleophile and favors the reaction with hard Lewis acids. Besides the nucleophilic properties of the phenolic hydroxy group, the electron-rich nature of the phenyl ring has been exploited for tyrosine-selective bioconjugation reactions, e.g., with in situ generated Mannich reagents,(308) diazonium salts,(309) or via ene-like reactions with triazoline diones.(310,311)
5.1. Tyrosine Targeting with Sulfur (VI) Fluorides
Because the inherent reactivity of tyrosine and lysine toward sulfur (VI) fluorides has already been discussed extensively in section 4.2, only application examples are provided in this section. Sulfonyl fluoride warheads have recently been used by Lyn Jones and colleagues from Pfizer to address the mRNA-decapping scavenger enzyme DcpS.(307) In a structure-based design approach, they modified known diaminoquinazoline-derived DcpS inhibitors to covalently address two proximal tyrosines, Tyr113 and 143, or the neighboring Lys142. The generated inhibitors featured low picomolar IC50 values in an ELISA-based DcpS activity assay (no binding kinetics were provided) and were approximately 2 orders of magnitude more potent than the reversible parent compound D153249 (123, Figure 51). X-ray and MS experiments showed that the ortho- and meta-substituted inhibitors 124a,b selectively modify Tyr113, while compound 124c with the CRG in the para-position reacts exclusively with Tyr143 (Figure 52). However, all of these inhibitors left Lys142 and His139 untouched, indicating that precise positioning of the warhead in conjunction with the appropriate intrinsic reactivity of the sulfonyl fluoride group for tyrosine confers specificity. It is worth mentioning that an analogous inhibitor with a silent click-tag revealed some off-targets that were not further disclosed.
Figure 51
Figure 51. Design of tyrosine-targeted sulfonyl fluorides as DcpS inhibitors.
Figure 52
Figure 52. Distinct binding modes of 124a–c in the respective X-ray crystal structures in complex with DcpS. The ligand is completely embedded in the protein environment, and the 2,4-diaminquinazoline core adopts a similar orientation in all structures. Conserved interactions include two hydrogen bonds of the quinazoline 2-amino group to the carboxylate of Glu185 and the backbone carbonyl of Pro204 as well as a hydrogen bond between the quinazoline 4-amino group and the Asp205 side chain. The second proton of the quinazoline 4-amine forms an intramolecular H-bond to the ether linker. The (protonated) quinazoline N1-atom is also hydrogen-bonded to Glu185. (A) ortho-Substituted derivative 124a covalently bound to Tyr113 (PDB 4QDE). The phenyl ring points to the “bottom” of the binding pocket. Additional hydrogen bonds are formed to the side chains of Lys142 and Tyr273. (B) meta-Substituted derivative 124b covalently bound to Tyr113 (PDB 4QEB). Covalent attachment is enabled by an “upward” orientation of the phenyl ring, giving rise to an additional hydrogen bond between the sulfonyl group and the Tyr143 side chain. (C) para-Substituted derivative 124c covalently bound to Tyr143 (PDB 4QDV). The overall orientation resembles that of 124a, but Tyr143 is labeled instead of Tyr113. No hydrogen bonds with Lys142, Tyr273 and His139 are observed in (B) and (C), and the latter two residues were omitted for clarity. No covalent modification of the proximal nucleophiles Lys142 and His139 was observed in any of the experiments.
In a very recent study, Nathanael Gray and co-workers made use of a sulfonyl fluoride warhead to generate SRPKIN-1 (126, Figure 53), the first targeted kinase ligand covalently addressing a tyrosine residue.(312) While screening an internal compound library in the cellular KiNativ profiling assay,(313) the approved anaplastic lymphoma kinase (ALK) inhibitor alectinib (125)(314) was found to exhibit strong inhibitory activity toward the SR-protein kinase SRPK1 (IC50 = 11 nM on isolated SRPK1). A cocrystal structure gave insight into the binding mode of alectinib in complex with SRPK1, suggesting that replacement of the 4-morpholinopiperidine moiety by a meta-substituted phenyl ring bearing a sulfonyl fluoride warhead might enable covalent targeting of a unique tyrosine (Tyr227) adjacent to the solvent-exposed front region of the ATP binding site. Installation of this headgroup conserved most of the SRPK1 activity (IC50 = 36 nM) while rendering the compound a less potent ALK inhibitor (IC50 = 195 nM). No kinetic data was provided, thus the contribution of the covalent interaction to the observed potency remains unclear. Cellular KiNativ profiling demonstrated selectivity for SRPK1 and SRPK2, and washout experiments supported the anticipated covalent binding mode. Labeling of Tyr227 was subsequently confirmed by MS but no X-ray crystal structure was published. Notably, the compound suppressed neovascularization in a choroidal neovascularization model (CNV) after intravitreal injection in mice.
Figure 53
Figure 53. Design of sulfonyl fluoride SRPKIN-1, the first tyrosine-targeted covalent kinase inhibitor.
The less reactive aryl fluorosulfates have been suggested as privileged tyrosine-targeted electrophiles as they preferably react with phenolic hydroxy groups when compared to free alkanols, amines, thiols, guanidines, and heterocyclic NH groups.(315) Interesting work highlighting the special properties of aryl fluorosulfate warheads was published in 2016 by Kelly, Sharpless, Wilson, and colleagues. They showed that structurally simple aryl fluorosulfates can react specifically with tyrosine side chains in certain intracellular lipid binding proteins.(268) By using a clickable PEGylated phenyl fluorosulfate (127, Figure 54), it was demonstrated that this compound class selectively (and slowly) labels relatively few proteins as contrasted by an analogous sulfonyl fluoride. The cellular retinoic acid binding protein (CRABP) 2 was identified as the key target of 127 in HeLa cell proteomes. Rudimentary optimization furnished biphenyl-derived analogue 128 and fluorescent probe 129, enabling higher (but still moderate) CRABP2 labeling rates (kinact = 0.106 min–1 for 129). Structural comparison of CRABP2 with other lipid-binding proteins targeted by 127 suggested a spatially conserved Arg–Arg–Tyr carboxylic acid binding motif to be the key for covalent modification. Intriguingly, mutational depletion of either of the arginine residues impaired labeling. Studies at variable pH values indicated that the arginine side chain lowers the pKa of the tyrosine phenol moiety, and a simultaneous stabilization of the fluoride leaving group is likely. Cell-permeable probe 128 showed a very low background proteome labeling in HEK293T cells. An X-ray crystal structure in complex with CRABP2 (Figure 55
Figure 54
Figure 54. Aryl fluorosulfate probes targeting CRABP2 used in chemical proteomics studies.
Figure 55
Figure 55. X-ray crystal structure of the fluorosulfate-based ligand 128 covalently bound to Tyr134 in CRABP2 (PDB 5HZQ). The ligand is deeply buried in the binding site, and the sulfate group is engaged in a direct hydrogen bond to the Arg132 side chain and water-mediated hydrogen bonds to the Arg111 side chain. The PEG-linker is not resolved and a second, slightly deviating conformation of the ligand and the Tyr134 side chain was omitted for clarity.
Subsequently, the same groups reported on a strategy they denominated ″inverse drug discovery″, where the highly specific nature of latent fluorosulfate electrophiles was utilized to identify proteins with hotspots activating the latter for covalent lysine or tyrosine binding.(269) By capitalizing on three distinct alkyne-labeled probes of intermediate complexity (130–132, Figure 56) in conjunction with unlabeled competitors, quantitative proteomics were employed to identify proteins that are efficiently labeled. In this approach, only a very low number of proteins was retrieved. This can be attributed to the very low reactivity of aryl fluorosulfates, however, the susceptibility of the reaction products to hydrolysis and elimination (vide infra), or the reaction with thiols to form unstable thiosulfate-S-esters (compare section 4.2) might also hamper product detection. Labeling at a specific site was validated for 11 targets by MS, mutagenesis, and X-ray crystallography using the recombinant proteins. As expected, covalent modification was only observed at tyrosine and lysine residues. Interestingly, probe 131 reacted with a largely different set of proteins compared to probes 130 and 132. Although no further optimization was performed, this study identified interesting targets that might be covalently addressed by sulfur (VI) chemistry. It should not remain unmentioned that the labeling sites could be reliably predicted by covalent docking which might be interesting for future drug discovery efforts.
Figure 56
Figure 56. Alkyne-tagged aryl fluorosulfate-based probes used in an “inverse drug discovery” approach.
5.2. Tyrosine Targeting by SNAr Reactions
LAS17 (133, Figure 57), an inhibitor with a 4,6-dichloro-1,3,5-triazine warhead, was recently shown by Lisa Crawford and Eranthie Weerapana to target Tyr108 in glutathione S-transferase π (GSTP1).(317) After having discovered by chemical proteomics that dichlorotriazines preferably label lysines while p-chloronitrobenzenes favor cysteines (see section 2.6),(130) a library of 20 N,N-disubstituted 4,6-dichloro-1,3,5-triazin-2-amines equipped with a click handle was synthesized and tested against HeLa cells at 1 μM. Protein enrichment revealed that one compound, LAS17, selectively modified a 25 kDa protein that was identified as GSTP1. This selectivity is quite surprising because 4,6-dichloro-1,3,5-triazin-2-amines chemically react with amino nucleophiles even at ambient temperature.(318) LAS17 inhibited GSTP1 in a concentration-dependent manner and activity was not comprised in the presence of the protein background from HeLa cell lysates. The second-order rate constant of inactivation (kinact/Ki) was determined as 3.12 × 104 M–1 s–1, and intact protein MS proved selective monolabeling. Further MS studies revealed Tyr108 as the only site of modification despite the presence of two highly reactive cysteine residues in GSTP1. It cannot be fully excluded that the reaction proceeds via an initial reaction with one of the more nucleophilic cysteines followed by transfer on Tyr108 to form the thermodynamically favored product. However, due to the distance between these residues (≥11 Å between the cysteines’ sulfur atoms and the phenolic oxygen in PDB 5X79) and their arrangement, such a low energy transfer pathway seems unlikely in this case. Mutation of Tyr108 to Phe prevented covalent modification and LAS17 possessed negligible activity on the mutant protein. It should be mentioned at this point that Tyr108 had previously been shown to be reactive toward sulfonyl fluoride-based probes.(319) Although this study highlights the ability of SNAr warheads to efficiently target tyrosine residues, it should be kept in mind that the employed electrophile features a high intrinsic reactivity. It is therefore remarkable that LAS17 only marginally labeled other proteins at a concentration of 1 μM emphasizing that intrinsic reactivity is not the only determinant for promiscuity and that the specificity and kinetics of reversible binding play a critical role for the selectivity of covalent inhibitors. In this context, further investigation of the specificity in cellular proteomes upon prolonged exposure to higher compound concentrations as well as a systematic evaluation of the minimal reactivity requirements for modifying Tyr108 with SNAr-type warheads would be very informative.
Figure 57
Figure 57. LAS17, a tyrosine-targeted dichlorotriazine-derived GSTP1 inhibitor.
6. Targeting Noncatalytic Serine and Threonine Residues
ARTICLE SECTIONS
Serine and threonine are abundant as the key catalytic residues in the active sites of proteases and other hydrolase enzymes. At these locations, the side chain hydroxy groups are activated by neighboring residues, e.g., in catalytic triads, dramatically increasing their nucleophilicity.(320) A plethora of covalent inhibitors has been developed for these enzymes, and as mentioned already, many of the CRGs described in this Perspective had initially been employed to target the active sites of hydrolases.(7,321) A comprehensive discussion of covalent inhibitors of this enzyme class, however, is far beyond the scope of this article.
In contrast, noncatalytic serine and threonine residues are usually poorly acidic and therefore hardly reactive. Targeting these amino acids is still a significant challenge in bioconjugation chemistry. There is a very prominent example for the covalent modification of a noncatalytic serine by a drug, namely the acetylation of Ser530 in cyclooxygenases by aspirin.(322) Nevertheless, reports on compounds targeting noncatalytic serine and threonine residues in a proximity-driven manner remain very rare. In general, hard Lewis acids, such as sulfur (VI) fluorides or oxophilic phosphorus (V) compounds but also boron-based reagents, may be suited best for addressing such hydroxy groups.
6.1. Targeting Noncatalytic Serine by Fluorosulfates
In a follow-up of the study on diaminoquinazolines targeting tyrosine residues in DcpS (see section 5.1, Figure 51), Lyn Jones and co-workers sought to reduce off-target labeling by replacing the sulfonyl fluoride warheads by less reactive fluorosulfates.(316) Key compound FS-p1 (134a, Figure 58) featured increased stability toward hydrolysis and adequate membrane permeability. The turnover of this inhibitor in human liver microsomes was even lower as for the unsubstituted parent compound DAQ1 (134b), albeit at the expense of potency (IC50 = 3.2 nM) compared to the corresponding sulfonyl fluoride 124c (IC50 = <0.02 nM, cf. Figure 51). Unexpectedly, however, compound 134a modified neither of the expected tyrosine residues nor the adjacent lysine. Instead, reaction with the noncatalytic Ser272 was observed in peptide mapping experiments. LC-MS studies using the full protein and MS-based analysis after tryptic digestion did not reveal the labeled protein as the predominant species but the dehydroalanine (Dha) elimination product (see the mechanism in Figure 58B). In contrast, native ESI-MS(323) detected the labeled protein along with minor amounts of the dehydroalanine product, which slowly increased over time, suggesting that the latter species is mainly an artifact and not dominant in the protein’s binding site on a short time scale. The covalent engagement of Ser272 was especially surprising because this residue is not functionally relevant as the S272A mutant retains activity. The authors argued that the additional oxygen spacer would position the sulfur atom in an unfavorable position for being attacked by one of the more nucleophilic tyrosine or lysine side chains. The positive electrostatic nature of the binding pocket featuring an adjacent histidine triad may further depress the pKa of the serine and/or facilitate the departure of the fluoride leaving group. One implication of the limited stability of the sulfate diester products is that classical strategies for monitoring (off)-target modification, e.g., the click-chemistry based capturing approaches discussed before, would not be ideal for this warhead type. Alternative strategies will be required to examine the full scope of protein modification by such compounds in proteomes. Further investigation will also be required to show whether the formation of dehydroalanine-containing proteins could be a safety issue in vivo, as these are electrophilic themselves and may react with other physiological nucleophiles to generate hapten-carrier adducts. On the other hand, the underlying elimination reaction might be optimized and serve as a handle for specific protein derivatization.
Figure 58
Figure 58. (A) Aryl fluorosulfate-based inhibitor FS-p1 targeting Ser272 in DcpS. (B) Proposed mechanism for the formation of the dehydroalanine elimination product.
7. Targeting Glutamate and Aspartate Side Chains
ARTICLE SECTIONS
Covalent modification of carboxylate residues as present in aspartate and glutamate side chains poses a special challenge due to the weakly nucleophilic nature of the (solvated) carboxylate group. Although carboxylates can react with many of the electrophiles discussed before (e.g., with epoxides or α-haloacetamides), these reactions are not specific to carboxylates and stronger nucleophiles react preferentially. Some approaches for the selective covalent trapping of carboxylates in proteins such as the photoclick-reaction with tetrazole-based reagents(324,325) or the reaction with α-diazo carboxamides(326) have been described as methods for bioconjugation. Cyclitol epoxides and aziridines as well as fluorinated glycosides have further found application as carboxylate-targeted electrophiles in activity-based probes and inhibitors of retaining glycosidases, an enzyme class featuring aspartate or glutamate residues as the catalytic nucleophile.(165) Sulfonate esters were shown to react preferentially with aspartate and glutamate in proteomes, but this behavior seems to be mediated by the respective protein environments as no such selectivity could be observed in solution.(157) On the other hand, it was shown that aziridines and stabilized diazo groups react with N-Boc-aspartate and benzoic acid in solution, but these CRGs failed to significantly label Asp12 in the K-Ras G12D mutant.(182) Boronic acids have further been suggested to interact with an aspartate in the front pocket of the EGFR kinase domain via reversible covalent bond formation,(327) but this interaction was not confirmed experimentally and is not specific for carboxylates. Hence, biocompatible warheads with a specific intrinsic reactivity for carboxylates are largely underdeveloped. However, selective and tunable CRGs for addressing this functionality would be particularly desirable because Asp and Glu constitute more than 10% of the protein sequence space(59) and are frequently found in or around binding pockets.
7.1. Targeting Carboxylates with Isoxazolium Salts
In a recent proof of concept study, Herbert Waldmann and colleagues employed isoxazolium salts derived from Woodward’s reagent K (WDK)(328) as site-specific covalent warheads for a glutamate residue in the lipoprotein binding chaperone phosphodiesterase (PDE) 6δ.(329) Because previous subnanomolar bis-sulfonamide-based inhibitors (exemplified by 135, Figure 59A) suffered from ligand displacement by allosteric release factor (ARL) 2/3 proteins,(330) covalent targeting was considered. Because no cysteine or lysine residues are available in the PDE6δ prenyl binding pocket, tosylate tags(331) were initially introduced for covalently addressing Glu88 but labeling efficiency was low. An alternative targeting strategy was inspired by Woodward’s isoxazolium-derived carboxylic acid activation reagents. N-Methyl isoxazolium salts (exemplified by general structure 136, Figure 59B) undergo ring opening in a base-promoted manner furnishing ketenimides (137), which readily react with carboxylates to form O-acylated 1,2-enolized β-ketoamides (138). The latter slowly rearrange to form the corresponding β-enol esters (139). It was hypothesized that initial ring opening could be initiated by proximal basic groups in the binding pocket, furnishing the reactive ketenimide species. Starting from 135, the benzyl moiety was replaced by a 4-unsubstituted N-methyl isoxazolium warhead attached via an ethylene spacer at the C5-position of the isoxazole ring (140a). The piperidine substituent was further replaced by a cyclohexyl ring in this series. As predicted, compound 140a and its homologue 140b covalently labeled PDE6δ at Glu88 as shown by mass spectrometry; covalent inactivation, however, was incomplete (80% after 30 min) and PDE6δ got reactivated within 24 h due to the limited stability of the reaction products. Shifting the attachment point to the C4-position of the isoxazole ring and introducing an additional methyl substituent at the C5-position (compounds 140c and 140d) increased labeling efficiency (>95% in less than 10 min) and only marginal cleavage was observed within 24 h. Finally, reintroduction of the piperidine nitrogen atom furnished 141, another promising compound from this series. Interestingly, removing the (piperidin-4-yl)methyl substituent from the sulfonamide nitrogen atom yielded a compound which did not effectuate any labeling pinpointing the critical role of reversible binding and accurate warhead positioning. Compound 140d possessed low reactivity toward the side chains of protected lysine and serine but cross-reacted with cysteine. However, no labeling of the PDE6δ E88A mutant was observed. As indicated by MS and fluorescence quenching studies, ARL proteins were unable to release the covalent ligands from the protein. Covalent attachment to Glu88 was further demonstrated by an X-ray crystal structure (Figure 60). It is worth noting that the ligand appears to be predominantly bound in the less stable form (compare structure 138). However, the electron density of the covalent tether is slightly less pronounced as for the rest of the ligand which might be attributed to partial conversion to the more stable β-enol ester product. Investigation of the selectivity of compound 141 using a cellular thermal shift assay (CETSA)(332,333) revealed only three off-targets (GDPGP1, PSMG3, PTGES2) along with PDE6δ.
Figure 59
Figure 59. Glutamate-targeted PDE6δ inhibitors. (A) Attachment of N-methyl isoxazolium warheads to reversible inhibitors exemplified by 135. (B) Mechanism of the reaction between the N-methyl isoxazolium group and carboxylates.
Figure 60
Figure 60. X-ray crystal structure of compound 140d covalently bound to Glu88 of PDE6δ (PDB 5NAL). The ligand is predominantly bound in the less stable O-acylated form and deeply buried in the binding site. Hydrogen bonds are formed by both oxygen atoms of the first sulfonyl group to the side chains of Arg61 and Gln78. An additional hydrogen bond is established between the second sulfonyl group and the side chain of Tyr149 (omitted for clarity).
The chemical stability of the conjugate against a 50-fold excess of hydroxylamine(334) suggests that the binding pocket shields the enol ester, thereby preventing the expected cleavage. It is likely that binding to surface-exposed carboxylate residues would be transient, and cleavage can also be expected after proteolytic degradation of the target protein, potentially reducing side effects by off-target binding and haptenization. However, whether these CRGs can be applied in living organisms remains to be proved. A general problem for in vivo applications might be found in the chemical stability of the warhead, which is good in acidic solution but only moderate at pH 7.4 and low at pH 9. Although this could offer a kinetic selectivity advantage, limited hydrolytic stability might be hardly compatible with oral dosing and the comparably slow distribution in vivo. It remains to be seen if reactivity tuning by modulating steric bulk and electronic properties of the substituents could resolve these issues. Although previous isoxazolium-derived activity-based protein profiling (ABPP) probes from the same group were able to penetrate cells,(335) it needs to be further investigated to what extent these charged molecules can cross biological barriers and how they are affected by metabolism in vivo. Finally, it will be interesting to see if other peptide coupling reagents might be tuned to become suitable carboxylate-targeted warheads for application in chemical probes or ultimately in drug discovery.
8. Targeting the Histidine Side Chain
ARTICLE SECTIONS
Although the histidine imidazole group is frequently mentioned as a potential nucleophile for covalent targeting, only limited rational efforts have been described to address this amino acid. Histidine residues react with sulfonyl fluorides to form readily hydrolyzable sulfonylimidazoles as shown by the covalent modification of His130 in the active site of Salmonella typhimurium ribose-phosphate diphosphokinase by 5′-FSBA (102, Figure 42).(336) Moreover, histidine residues can undergo aza-Michael addition reactions with α,β-unsaturated ketones or aldehydes. For example, histidine residues were shown to react with 4-hydroxynonenal(337) or prostaglandin J2,(338) and more recently, a histidine of the vitamin D receptor has been covalently addressed with vitamin D-derived enones.(339)
8.1. Alkylation of Histidine by Spiro-epoxides
One of the prominent compounds addressing the histidine side chain is the natural product fumagillin (143, Figure 61) and the derived former clinical candidate beloranib (142), which bind to one of the active site histidines in methionine aminopeptidase (MetAP) 2 via opening of a spiro-epoxide (Figure 61B).(340) To improve the poor pharmacokinetics of this substance class, Aubry Miller and colleagues designed spiro-epoxytriazoles as drug-like fumagillin analogues (Figure 61C, general structures 144 and 145).(341) Several potent inhibitors of human MetAP2 were generated with key compound 145a (Figure 61D), featuring an IC50 value of 220 nM while its stereoisomers were inactive. As expected, enzyme inhibition was time-dependent and correlated well with cellular activities in human umbilical vein endothelial (HUVE) and HT1080 cells. Although the keto and benzoyl derivatives possessed poor stability in plasma and mouse liver microsomes, carbamate analogues were significantly more stable, even when compared with the former drug candidate beloranib. The labeling of His231 was confirmed by X-ray crystallography (Figure 62), while solvent-exposed nucleophiles (such as Cys290 and Lys427) remained untouched. These results indicate that histidine residues can be addressed specifically with relatively weak electrophiles, making them suitable targets for covalent inhibitor design. However, as for other moderate nucleophiles, the ligandability may largely depend on the precise positioning of the CRG and the target residues distinct nucleophilicity and thus on the surrounding protein environment.
Figure 61
Figure 61. Spiro-epoxides as histidine-targeted covalent inhibitors of hMetAP2. (A) Former drug candidate beloranib. (B) Reaction of the natural product fumagillin with His231 in hMetAP2. (C) Simplified fumagillin-derived structures. (D) hMetAP2 inhibitor 145a with improved PK properties.
Figure 62
Figure 62. X-ray crystal structure of the covalent complex between hMetAP2 and compound 145a (PDB 5CLS). His231 is covalently attached to the methylene group formed from the terminal carbon atom of the epoxide ring. The ensuing hydroxy group is linked to Asp251 and His382 by water-mediated hydrogen bonds. A direct hydrogen bond between the carbamate’s carbonyl group and the Asn329 backbone NH, and further water-mediated hydrogen bonds additionally anchor the ligand in the binding site.
8.2. Reversible Addition of Histidine to α-Cyanoenones
A covalent-reversible approach was recently pursued by Clarissa Jakob and co-workers from AbbVie to address a histidine in wild-type isocitrate dehydrogenase (IDH)1.(342) A high-throughput screen identified inhibitor 146a (Figure 63A) with an IC50 value of 410 nM. Optimization furnished the most potent carboxy-substituted analogue 146b (IC50 = 41 nM) and the slightly less active but cell permeable key compound 146c (IC50 = 110 nM). The latter inhibitor decreased reductive glutaminolysis in A498 cells in a dose-dependent manner, suggesting cellular target occupancy. Covalent modification of His315 in the NADPH binding pocket via aza-Michael addition to the α-cyanoenone moiety was confirmed by X-ray crystallography (Figure 63B), while no concomitant modification of exposed cysteines was observed. In accordance with the X-ray crystal structure showing a hydrogen bond between the backbone of Ser326 and the nitrile group, the removal of the α-cyano substituent in series 146 was detrimental to activity, while saturation of the double bond precluding covalent modification led to a moderate 16-fold loss in inhibitory potency. The latter finding suggests that the hydrogen bond to the nitrile group not only increases the inhibitors electrophilicity but is also crucial as a noncovalent key recognition element. Reversibility was demonstrated by wash out and jump dilution experiments. Consistent with transient covalent binding, no adducts were observed in mass spectrometric experiments.
Figure 63
Figure 63. α-Cyanoenones as histidine-targeted covalent-reversible IDH1 inhibitors. (A) Hit compound 146a and optimized derivatives. (B) X-ray crystal structure of 146c bound to IDH1 (PDB 6BL1). His315 is covalently attached to the β-position of the enone precursor. A key hydrogen bond is formed between the α-cyano moiety and the backbone NH of Se326. The enone keto group is hydrogen-bonded to the Lys374 side chain, while the diarylamino group forms a charge-assisted hydrogen bond to the carboxylate of Asp375.
9. Targeting Methionine Side Chains
ARTICLE SECTIONS
Methionine is among the rarest amino acids in vertebrates and due to its high lipophilicity, typically buried within proteins. As mentioned before, the nucleophilicity of the methionine side chain is only moderate, however, its sulfur atom can be readily oxidized. Protocols for methionine labeling typically rely on highly reactive electrophiles at low pH.(343)
9.1. Redox-Activated Labeling of Methionine by Oxaziridines
An interesting new method for the selective labeling of methionine residues in proteins and cell lysates has recently been identified by the groups of Dean Toste and Christopher Chang.(343) They aimed to exploit the distinct redox activity of methionine for redox-activated chemical tagging (ReACT) under physiological conditions. A screening identified oxaziridines as suitable strain-driven sulfur imidation reagents.(344) Incorporating the oxaziridine nitrogen atom into a weakly electron-withdrawing urea moiety (exemplified by compound 147a, Figure 64A) avoided problems with concomitant sulfoxide formation observed for analogous carbamates (e.g., compound 147b). Sulfur imidation proceeds via a nucleophilic attack of the sulfur lone pair at the oxaziridine nitrogen atom followed by ring opening and release of benzylic aldehydes or ketones to furnish the S-imidation product (Figure 64B/C). The S-oxidation byproduct is formed in an analogous manner by attack at the oxygen atom. Labeling was selective for methionine over other amino acids (free cysteine and selenocysteine were oxidized to their cystine forms), and the products were relatively stable even in the presence of acid, base, and reducing agents. Alkynylated probe 147c at a 1 mM concentration incubated for 10 min with HeLa cell lysates labeled 235 methionine residues, and only a single lysine residue. Different concentrations of 147c showed a dose-dependent increase in the number of labeled residues allowing the identification of hyper-reactive methionines. The suitability of the approach for selective protein labeling and the construction of antibody–drug conjugates was also demonstrated. Despite their chemoselectivity, the presented oxaziridine-based probes are still highly reactive and convert methionine at rates similar to CuAAC reactions. It remains to be seen if steric shielding and/or tuning of electronic properties could furnish more specific warheads for chemical probe or drug design and if the bulkiness of such CRGs will limit their use with respect to the spatial constrains of many binding pockets. The underlying principle, however, is very promising and might stimulate research on similar approaches for specifically addressing methionine side chains.
Figure 64
Figure 64. Methionine-targeted oxaziridines. (A) Urea and carbamate-derived analogues. (B) Reaction with methionine via S-imidation or concomitant S-oxidation. Conditions: (1) D2O/CD3OD = 1:1, 2.5 min or (2) D2O/CD3OD = 95:5, 20 min. (C) Reaction mechanism of covalent methionine modification.
10. Targeting Other Amino Acids
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It is not surprising that relatively few methods have been described to selectively address weak nucleophiles such as arginine, asparagine, glutamine, or tryptophan. For example, the bidentate nature of arginine’s guanidinium group has been exploited for reactions with glyoxal-derived reagents,(345) forming comparably stable cyclic products while the reaction with lysine or cysteine is highly reversible. However, the latter compound class is unlikely to find broad application in drug discovery due to metabolic liability and potential toxicity issues. A recent method has been described for metal-free tryptophan-selective bioconjugation in proteins.(346) Although being conceptually interesting, this method requires an organoradical reagent and sodium nitrite as an additive precluding applications in TCI design. Addressing phenylalanine or amino acids with nonactivated aliphatic side chains is even more challenging. Although catalysis-based approaches might be practicable for bioconjugation in vitro, such chemistries are difficult to realize in cells or even in vivo. Therefore, it is unlikely that these amino acids will be amenable to covalent targeting in a medicinal chemistry setting in the near future.
11. Summary and Perspective
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Covalent targeting has become an extremely powerful tool in drug discovery and chemical biology and substantial effort has recently been put into developing or repurposing warheads for TCI design. An overview of the warhead classes discussed in this article and their key characteristics is provided in Table 2. The reader is referred to the respective sections and the literature cited therein for details.
Table 2. Overview of the Warhead Classes Discussed in This Article
a
Excluding catalytic nucleophiles. Data based on the studies discussed in the respective chapters.
b
L: Protein/peptide/amino acid (labeling or reactivity assay);. P: Protein (activity, binding affinity, kinact/KI). Y: Cell lysate (chemical proteomics study). C: Intact cell (functional assay or chemical proteomics study). I: In vivo (mammals or human). Data based on the reports discussed in the respective sections and additional searches in PubMed and the DrugBank.(146)
c
Application restricted to the catalytic cysteine of DUBs and related cysteine proteases so far.
d
Modified in solution but aziridines were unable to address Asp12 in the K-Ras G12D mutant.
e
Suggested by experiments with n-butylamine as a model nucleophile.
f
Forms unstable S-ester reaction products with cysteine.
g
Hemithioacetal formation with Cys is likely but rapidly reversible.
Despite the plethora of CRGs described in the current literature, selecting the right warhead for a specific application remains a nontrivial task. Especially in vivo, factors to be considered reach far beyond the structure of the protein and the nature of the target amino acid and include properties like target turnover, tissue distribution, and (sub)-cellular location, among others. Metabolic stability and chemical reactivity of the ligand also need to be well balanced, and the ideal window largely depends on the projected application. In this context, it is important to note that extrahepatic clearance is a major determinant of the pharmacokinetics of common acrylamide-derived TCIs.(347) Oxidative metabolism via reactive epoxide intermediates is another well-known biotransformation of α,β-unsaturated amides.(348) However, little is known about the metabolic fate of many CRGs discussed here. Reactivity toward glutathione, but also other thiol-containing reagents, has frequently been employed as a surrogate parameter to describe the nonspecific reactivity toward biological nucleophiles. In cells, however, much of the GSH-conjugation is mediated by glutathione S-transferases. Consequently, GSH addition under real physiological conditions might be much faster than estimated by the common (enzyme-free) GSH binding assays. An additional level of complexity is added by the presence of hyper-reactive cysteine residues in various proteins that could potentially engage even weakly reactive electrophiles. Similarly, certain features of protein binding sites might activate individual CRGs to become more electrophilic. In the light of this complexity, it should not remain unmentioned that some of the electrophiles discussed in this Perspective have only been evaluated in isolated proteins or peptides so far. Although these chemotypes may become useful in TCI design, the potential of such CRGs remains unclear until extensive profiling of reactivity, specificity, and stability in cells or in vivo has been performed.
Warhead selection typically starts with the estimation of reactivity required with respect to the desired target amino acid. Although computational reactivity prediction for certain electrophiles has significantly advanced within the last years,(12,158,349) precise and comparable rating over different warhead classed and nucleophiles remains a major challenge. When sufficiently exposed and reactive cysteines are to be addressed and slow depletion by GSH or other thiols is not an issue, α,β-unsaturated amides generally represent a safe bet. However, problems may arise when the target residue is poorly reactive, difficult to access, or incompatible with the spatial and geometric requirements of this electrophilic headgroup. Here, alternative acceptors, such as alkenylated or alkynylated heteroarenes, might come into play. SNAr warheads may offer advantages if spatially defined targeting needs to be combined with good metabolic stability and a highly tunable reactivity, especially if some additional steric bulk is not a problem. In the best case scenario, electron-deficient (hetero)arenes, which are already present in many biologically active compounds may simply be equipped with suitable leaving groups for covalently addressing proximal nucleophilic amino acids. Carbocyclic strain release warheads, which have a very low literature precedence, may be valuable alternatives especially if IP claims are an issue. Despite their promising chemical properties, however, the latter moieties have only been evaluated against isolated peptides so far. Systematic implementation of metabolically labile CRGs, such as fumaric acid esters or epoxides, is an option where short time exposure could offer a kinetic selectivity advantage. If reversible cysteine targeting is desired, α-cyanoacrylamides and analogous dually activated Michael acceptors are an up-to-date option with clinically approved representatives. Activated nitriles such as cyanamides or 2-cyanopyri(mi)dines represent suitable alternatives that have shown promising in vivo properties and aldehydes may also be employed in some cases.
Although the above-mentioned CRGs are typically used for cysteine targeting, many of them can also react with weaker nucleophiles such as lysine, tyrosine, or histidine. As discussed before, selective engagement of these less reactive residues is more challenging. Harder electrophiles, e.g., sulfonyl fluorides or activated esters, are more suitable to react with amino or hydroxy bases. Vinyl sulfones and the corresponding sulfonamides have further been shown to react readily with the lysine ε-amino group. Although the latter CRGs may be used for targeting lysines and tyrosines, most of these moieties have certain liabilities such as high intrinsic reactivity (vinyl sulfones and sulfonyl fluorides), limited stability toward hydrolysis (sulfonyl fluorides), and susceptibility to metabolic inactivation (activated esters). In the case of sulfur (VI) fluorides, the weakly reactive aryl fluorosulfates and the highly tunable sulfonimidoyl fluorides may address these issues and clearly merit further investigations. For reversible lysine targeting, 2-carbonylarylboronic acids forming stabilized Schiff bases may open up future avenues and further proof of concept, especially in vivo, would be highly desirable. However, despite these advances, the scope of lysine and tyrosine-targeted warheads is still limited and studies addressing the histidine side chain are even less abundant in the recent literature. Consequently, there is an urgent need for the development of novel CRGs which reliably and specifically address these residues without cross-reacting with cysteines.
Many weakly nucleophilic (e.g., glutamine, asparagine, arginine as well as nonactivated serine or threonine) or electrophilic (e.g., tryptophan) amino acids cannot be targeted reliably with the current warhead chemistry, necessitating further research to expand our scope of ligandable residues. Encouragingly, recent studies on glutamate/aspartate or methionine targeted CRGs hold the promise that at least some of the challenges associated with weaker nucleophiles could be addressed in the near future. Furthermore, the recognition of the role of cysteine oxidation in redox regulation has stimulated promising research on nucleophilic CRGs targeting electrophilic sulfenic acids and sulfenamides, which represent transient key oxidation products of cysteines’ thiols. Nevertheless, these concepts still have to prove their applicability in drug discovery and further refinement or alternative chemical approaches will be necessary when aiming for clinical applications.
Likewise, many issues with the more established warheads remain to be resolved. For example, none of the presented studies on sulfonyl fluorides, SNAr electrophiles, or activated esters investigated the toxic potential of the leaving group. Depending on the dose and the abundance/distribution of the target protein, it is conceivable that high local fluoride concentrations may result from fluoride-containing warheads in vivo. Similarly, phenols and other alcohols eliminated from activated esters could possess a distinct biological activity or toxicity themselves. Although these factors are not major issues for many chemical biology applications, they necessitate a detailed assessment prior to utilization in humans.
As it can be noticed throughout this article, IC50 values still dominate the literature on covalent ligands although the more laborious determination of binding kinetics is becoming more common. This reliance on IC50 data has implications on covalent ligand design because the observed differences in potency can be driven by reversible binding (KI) but also by the efficiency of covalent bond formation (kinact). In this context, it is interesting to note that the diffusion-based upper limit for the specificity constant (kcat/KM) of kinetically perfect enzymes is in the range of 108−109 M−1 s−1. Specific ligand association (described by kon), which is retarded, for example, by the requirement for pre-orientation or by induced fit, is usually slower and cannot exceed this cap.(34,350) Naturally, the same considerations apply to kinact/KI.
A well-designed TCI should be a potent and specific reversible binder to allow for selective target modification at low inhibitor concentrations. It should place the reactive moieties in favorable positions in terms of distance and angle. Similarly, the orientation toward activating residues in the binding cleft can play a role. The latter factors are crucial to enable a rapid bond formation between the reaction partners. The proper alignment of the reactive residues becomes especially important when low reactivity warheads are employed because the key properties of covalent binders (see the Introduction) would be lost with inactivation kinetics becoming too slow. In such a case, increased reversible potency could easily be mistaken as a result of covalent binding. Remarkably, recent data suggest that the efficacy of approved covalent EGFR inhibitors relies predominantly on reversible interactions.(219) Consequently, understanding binding kinetics is key to generate a properly balanced TCI which benefits from both the reversible and the covalent binding event.
To determine the specificity of novel CRGs and the derived TCIs in cells or even tissues, robust and convenient assays are required. Fortunately, considerable advances in chemical proteomics have made the assessment of off-targets in cellular proteomes becoming more and more common. Proteomic approaches provide very powerful tools to determine the fate of reactive ligands. Nevertheless, they also have their limitations. Methods employing tagged ligands to enrich covalently bound proteins, for example, may ignore the influence of the tag on ligand binding. On the other hand, approaches exploiting the differential labeling pattern of promiscuous reactive probes in the presence of a competitor ligand capture only a fraction of all potentially reactive amino acids present (e.g., the entire “cysteinome” or the “lysinome”) suggesting that relevant reaction sites could be missed. Even more of an issue are the intrinsic limitations of reactivity-based methods in identifying noncovalent interactions, which can significantly contribute to the biological profiles of reactive ligands. Many other issues are known and a further discussion may be found elsewhere.(351) Combination with emerging techniques, e.g., cellular thermal shift assays (CETSA)(332) or thermal proteome profiling,(333) and the ever increasing number of biochemical and cellular screening platforms, provides an opportunity to obtain a more complete picture. In this context, it is worth mentioning that little effort has been made so far to investigate the binding of reactive ligands to nonprotein off-targets, for example, the nucleobases in RNA and DNA. Such investigations would become even more important when employing more versatile warhead chemistries because each functional group features a distinct reactivity toward these nucleophiles. Altogether, understanding the fate of reactive ligands in complex living organisms still represents a fascinating challenge and a continuously developing area of research.
Ultimately, it seems unlikely that alternative warheads will replace cysteine-targeted α,β-unsaturated amides as the dominant chemical species in TCI design in the near future. Nevertheless, α,β-unsaturated amides are still far from being perfect warheads and do often not provide the general and predictable solutions they promise. Therefore, the CRGs presented here offer a valuable toolbox for the manifold applications where cysteine targeting with α,β-unsaturated amides cannot satisfy all needs or cases where simply no cysteine residues are available for covalent modification. Finally, we can expect a broadening of the scope of CRGs employed in drug discovery as challenging targets require the deliberate use of tailor-made warheads for success.
The authors declare no competing financial interest.
Author Information
ARTICLE SECTIONS
- Matthias Gehringer - Department of Pharmaceutical/Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany;
http://orcid.org/0000-0003-0163-3419;
- Stefan A. Laufer - Department of Pharmaceutical/Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany;http://orcid.org/0000-0001-6952-1486
The authors declare no competing financial interest.
Biographies
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Matthias Gehringer
Matthias Gehringer studied chemistry at the Karlsruhe Institute of Technology, the Ecole Nationale Supérieure de Chimie de Montpellier, and the University of Heidelberg, and obtained his Ph.D. from Tübingen University working on reversible and irreversible kinase inhibitors. As a postdoctoral researcher at the Swiss Federal Institute of Technology (ETH) Zürich, he focused on the total synthesis of mycolactones and on targeted antibiotic–protein conjugates. He recently returned to Tübingen, where he is currently establishing an independent research group. His research interests include medicinal chemistry, chemical biology, natural-product synthesis, and innovative drug targeting approaches.
Stefan A. Laufer
Stefan A. Laufer studied Pharmacy and completed his Ph.D. from Regensburg University. After postdoctoral research in Frankfurt, he took a position in the pharmaceutical industry but maintained lectureships at Frankfurt and later at Mainz University, where he finished his habilitation in 1997. Since 1999, he has been full professor (chair) for pharmaceutical and medicinal chemistry at Tübingen University. He is cofounder/spokesman of ICEPHA (Interfaculty Center for Pharmacogenomics and Pharma Research), TüCADD (Tübingen Center for Academic Drug Discovery), and cofounder of the two startups CAIR Biosciences and Heparegenix. Three compounds from his lab made it first into man. He is currently (2016–2019) president of the German Pharmaceutical Society (DPhG). His research interests are protein kinase inhibitors and eicosanoid modulators.
Acknowledgments
ARTICLE SECTIONS
We thank Dr. Michael Forster and Dr. Marcel Günther for fruitful discussions and Dr. Apirat Chaikuad for scientific advice. Kristine Schmidt, Dr. Michael Forster, Dr. Marcel Günther, and Bent Präfke are gratefully acknowledged for proof-reading. We thank Valentin Wydra and Nathanael Disch for assistance in the preparation of the manuscript and the TOC graphic. M.G. gratefully acknowledges financial support by the Institutional Strategy of the University of Tübingen (Deutsche Forschungsgemeinschaft, ZUK 63) and the Postdoctoral Fellowship Programme of the Baden-Württemberg Stiftung.
| Abbreviations Used | |
| ABPP | activity-based protein profiling |
| AChE | acetylcholinesterase |
| ADME | absorption, distribution, metabolism and excretion |
| ALDH2 | aldehyde dehydrogenase 2 |
| ALK | anaplastic lymphoma kinase |
| AR | androgen receptor |
| ARL | allosteric release factor |
| BTK | Bruton’s tyrosine kinase |
| CDK | cyclin-dependent kinase |
| CETSA | cellular thermal shift assay |
| CNV | choroidal neovascularization model |
| CRABP | cellular retinoic acid binding protein |
| CRG | covalent reactive group |
| CuAAC | copper(I)-catalyzed alkyne–azide cycloaddition |
| CYP | cytochrome P |
| DcpS | scavenger mRNA-decapping enzyme |
| DDAH | dimethylarginine dimethylaminohydrolase |
| DFT | density functional theory |
| Dha | dehydroalanine |
| DMF | dimethyl fumarate |
| DMPK | drug metabolism and pharmacokinetics |
| DMSO | dimethyl sulfoxide |
| DTT | dithiothreitol |
| DUBs | deubiquitinating isopeptidases |
| EC50 | half-maximum effective concentration |
| EGFR | epidermal growth factor receptor |
| ErbB | protein family of four receptor tyrosine kinases (ErbB/Her 1–4) |
| ESI-MS | electrospray ionization-mass spectrometry |
| FDA | Food and Drug Administration |
| FSBA | 5′-(4-fluorosulfonylbenzoyl)adenosine |
| FGFR | fibroblast growth factor receptor |
| FMK | fluoromethylketone |
| GAG | glycosaminoglycan |
| GK | gatekeeper |
| GPCR | G-protein coupled receptor |
| GPX | glutathione peroxidase |
| GSH | glutathione |
| GSTP1 | glutathione S-transferase π or P1 |
| H1975 | human lung-cell line |
| hCES | human carboxylesterase |
| HCV | hepatitis C virus |
| HDAC8 | histone deacetylase 8 |
| HEK293 | human embryonic kidney 293 cells |
| hERG | human ether-à-go-go-related gene |
| HSAB | hard and soft acids and bases |
| HTS | high throughput screening |
| HUVE | human umbilical vein endothelial |
| IC50 | half-maximum inhibitory concentration |
| ICL | isocitrate lyase |
| IDH | isocitrate dehydrogenase |
| IRE | inositol-requiring enzyme |
| ITAM | immunoreceptor tyrosine-based activation motif |
| ITK | interleukin-2-inducible T-cell kinase |
| JAK | Janus kinase |
| K-Ras | p21 GTPase (oncogen first found in Kirsten rat sarcoma virus) |
| MAO | monoamine oxidase |
| MetAP | methionine aminopeptidase |
| MGMT | (O6-)methylguanine-DNA-methyltransferase |
| MMF | monomethyl fumarate |
| MSF | methanesulfonyl fluoride |
| MSBT | 2-(methanesulfonyl)benzothiazole |
| MurA | UDP-N-acetylglucosamine enolpyruvyl transferase |
| NAC | N-acetylcysteine |
| NBD-dye | nitrobenzoxadiazole-dye |
| NCI | National Cancer Institute |
| NHS | N-hydroxysuccinimid |
| NMR | nuclear magnetic resonance |
| NS5B | nonstructural protein 5B |
| PDB | protein data bank |
| PDIA1 | protein disulfide isomerase A1 |
| PEG | polyethylene glycol |
| PEITC | phenethyl isothiocyanate |
| PGAM1 | phosphoglycerate mutase 1 |
| PLK | polo-like kinase |
| PLP | pyridoxal phosphate |
| PMBCs | peripheral blood mononuclear cells |
| PMSF | phenylmethane sulfonyl fluoride |
| PPARs | peroxisome proliferator-activated receptors |
| ReACT | redox-activated chemical tagging |
| ROS | reactive oxygen species |
| SAR | structure–activity relationship |
| SH | Scr homology |
| SNAr | nucleophilic aromatic substitution |
| SPR | surface plasmon resonance |
| SRC | steroid receptor coactivator |
| SRPK1 | SR-protein kinase 1 |
| SuFEx | sulfur (VI) fluoride exchange |
| SUMO | small ubiquitin-related modifier |
| TCEP | tris(2-carboxyethyl)phosphine |
| TCI | targeted covalent inhibitor |
| TM | transmembrane domain |
| TR | thyroid hormone receptor |
| TR-FRET | time-resolved Förster resonance energy transfer |
| TRPA1 | transient receptor potential cation channel A1 |
| Ub | ubiquitin |
| WDK | Woodward’s reagent K |
References
ARTICLE SECTIONS
This article references 351 other publications.
- 1Mann, M.; Jensen, O. N. Proteomic Analysis of Post-Translational Modifications. Nat. Biotechnol. 2003, 21 (3), 255– 261, DOI: 10.1038/nbt0303-255[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhsFajsro%253D&md5=ea9706baed997144456697914d190260Proteomic analysis of post-translational modificationsMann, Matthias; Jensen, Ole N.Nature Biotechnology (2003), 21 (3), 255-261CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)A review. Post-translational modifications modulate the activity of most eukaryote proteins. Anal. of these modifications presents formidable challenges but their detn. generates indispensable insight into biol. function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purifn. of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in mol. detail, novel mass spectrometric peptide sequencing and anal. technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.
- 2Schopfer, F. J.; Cipollina, C.; Freeman, B. A. Formation and Signaling Actions of Electrophilic Lipids. Chem. Rev. 2011, 111 (10), 5997– 6021, DOI: 10.1021/cr200131e[ACS Full Text
], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1SltrvI&md5=3fc9a6a520d8ca4dfc2ff3ad8545c160Formation and Signaling Actions of Electrophilic LipidsSchopfer, Francisco J.; Cipollina, Chiara; Freeman, Bruce A.Chemical Reviews (Washington, DC, United States) (2011), 111 (10), 5997-6021CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. This review addresses lipid-derived electrophilic species by first focusing on biochem. characteristics of electrophiles and the mechanisms underlying the endogenous formation of electrophilic species. This information is placed in the context of the cell signaling pathways modulated by electrophilic fatty acid derivs. and how these reactions can impact physiol. and pathophysiol. processes. - 3Allis, C. D.; Jenuwein, T. The Molecular Hallmarks of Epigenetic Control. Nat. Rev. Genet. 2016, 17 (8), 487– 500, DOI: 10.1038/nrg.2016.59[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGiur%252FJ&md5=dbbff4b5de8a0316f16664f73f3ad16dThe molecular hallmarks of epigenetic controlAllis, C. David; Jenuwein, ThomasNature Reviews Genetics (2016), 17 (8), 487-500CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)Over the past 20 years, breakthrough discoveries of chromatin-modifying enzymes and assocd. mechanisms that alter chromatin in response to physiol. or pathol. signals have transformed our knowledge of epigenetics from a collection of curious biol. phenomena to a functionally dissected research field. Here, we provide a personal perspective on the development of epigenetics, from its historical origins to what we define as 'the modern era of epigenetic research'. We primarily highlight key mol. mechanisms of and conceptual advances in epigenetic control that have changed our understanding of normal and perturbed development.
- 4Uetrecht, J. Idiosyncratic Drug Reactions: Current Understanding. Annu. Rev. Pharmacol. Toxicol. 2007, 47 (1), 513– 539, DOI: 10.1146/annurev.pharmtox.47.120505.105150[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXit1altb0%253D&md5=587b03e88f3b881c87d383818bea7f47Idiosyncratic drug reactions: current understandingUetrecht, JackAnnual Review of Pharmacology and Toxicology (2007), 47 (), 513-539CODEN: ARPTDI; ISSN:0362-1642. (Annual Reviews Inc.)A review. Clin. characteristics and circumstantial evidence suggest that idiosyncratic drug reactions are caused by reactive metabolites and are immune-mediated; however, there are few definitive data and there are likely exceptions. There are three principal hypotheses for how reactive metabolites might induce an immune-mediated idiosyncratic reaction: the hapten hypothesis, the danger hypothesis, and the PI hypothesis. It has been proposed that some idiosyncratic reactions, esp. those involving the liver, represent metabolic idiosyncrasy; however, there are even less data to support this hypothesis. The unpredictable nature of these reactions makes mechanistic studies difficult. There is a very strong assocn. with specific human leukocyte antigen (HLA) genes for certain reactions, but this has only been demonstrated for very few drugs. Animal models represent a very powerful tool for mechanistic studies, but the no. of valid models is also limited. There may be biomarkers of risk; however, much more work needs to be done.
- 5Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The Resurgence of Covalent Drugs. Nat. Rev. Drug Discovery 2011, 10 (4), 307– 317, DOI: 10.1038/nrd3410[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktVGmu7g%253D&md5=2190289081e151416c097be4a5b04460The resurgence of covalent drugsSingh, Juswinder; Petter, Russell C.; Baillie, Thomas A.; Whitty, AdrianNature Reviews Drug Discovery (2011), 10 (4), 307-317CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Covalent drugs haveproved to be successful therapies for various indications, but largely owing to safety concerns, they are rarely considered when initiating a target-directed drug discovery project. There is a need to reassess this important class of drugs, and to reconcile the discordance between the historic success of covalent drugs and the reluctance of most drug discovery teams to include them in their armamentarium. This Review surveys the prevalence and pharmacol. advantages of covalent drugs, discusses how potential risks and challenges may be addressed through innovative design, and presents the broad opportunities provided by targeted covalent inhibitors.
- 6Bauer, R. A. Covalent Inhibitors in Drug Discovery: From Accidental Discoveries to Avoided Liabilities and Designed Therapies. Drug Discovery Today 2015, 20 (9), 1061– 1073, DOI: 10.1016/j.drudis.2015.05.005[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptlOnu7s%253D&md5=6ba5b016d2804c7d56838a2724b9a0b9Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapiesBauer, Renato A.Drug Discovery Today (2015), 20 (9), 1061-1073CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)Drugs that covalently bond to their biol. targets have a long history in drug discovery. A look at drug approvals in recent years suggests that covalent drugs will continue to make impacts on human health for years to come. Although fraught with concerns about toxicity, the high potencies and prolonged effects achievable with covalent drugs may result in less-frequent drug dosing and in wide therapeutic margins for patients. Covalent inhibition can also dissoc. drug pharmacodynamics (PD) from pharmacokinetics (PK), which can result in desired drug efficacy for inhibitors that have short systemic exposure. Evidence suggests that there is a reduced risk for the development of resistance against covalent drugs, which is a major challenge in areas such as oncol. and infectious disease.
- 7Powers, J. C.; Asgian, J. L.; Ekici, Ö. D.; James, K. E. Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases. Chem. Rev. 2002, 102 (12), 4639– 4750, DOI: 10.1021/cr010182v[ACS Full Text], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XosFSnsr4%253D&md5=eb21ff0297b87537a7d459c92f13c75dIrreversible inhibitors of serine, cysteine, and threonine proteasesPowers, James C.; Asgian, Juliana L.; Ekici, Oezlem Dogan; James, Karen EllisChemical Reviews (Washington, DC, United States) (2002), 102 (12), 4639-4750CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Topics discussed include alkylating, acylating, phosphonylating, and sulfonylating agents.
- 8Bachovchin, D. A.; Cravatt, B. F. The Pharmacological Landscape and Therapeutic Potential of Serine Hydrolases. Nat. Rev. Drug Discovery 2012, 11 (1), 52– 68, DOI: 10.1038/nrd3620[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFOktA%253D%253D&md5=4fde1e23117ad11848a9c2eef85376b7The pharmacological landscape and therapeutic potential of serine hydrolasesBachovchin, Daniel A.; Cravatt, Benjamin F.Nature Reviews Drug Discovery (2012), 11 (1), 52-68CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Serine hydrolases perform crucial roles in many biol. processes, and several of these enzymes are targets of approved drugs for indications such as type 2 diabetes, Alzheimer's disease and infectious diseases. Despite this, most of the human serine hydrolases (of which there are more than 200) remain poorly characterized with respect to their physiol. substrates and functions, and the vast majority lack selective, in vivo-active inhibitors. Here, we review the current state of pharmacol. for mammalian serine hydrolases, including marketed drugs, compds. that are under clin. investigation and selective inhibitors emerging from academic probe development efforts. We also highlight recent methodol. advances that have accelerated the rate of inhibitor discovery and optimization for serine hydrolases, which we anticipate will aid in their biol. characterization and, in some cases, therapeutic validation.
- 9Paul, S. M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.; Lindborg, S. R.; Schacht, A. L. How to Improve R&D Productivity: The Pharmaceutical Industry’s Grand Challenge. Nat. Rev. Drug Discovery 2010, 9 (3), 203– 214, DOI: 10.1038/nrd3078[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitFemsbg%253D&md5=2f32bcc48c869290eef18ff9400afcc5How to improve R&D productivity: the pharmaceutical industry's grand challengePaul, Steven M.; Mytelka, Daniel S.; Dunwiddie, Christopher T.; Persinger, Charles C.; Munos, Bernard H.; Lindborg, Stacy R.; Schacht, Aaron L.Nature Reviews Drug Discovery (2010), 9 (3), 203-214CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. The pharmaceutical industry is under growing pressure from a range of environmental issues, including major losses of revenue owing to patent expirations, increasingly cost-constrained healthcare systems and more demanding regulatory requirements. In our view, the key to tackling the challenges such issues pose to both the future viability of the pharmaceutical industry and advances in healthcare is to substantially increase the no. and quality of innovative, cost-effective new medicines, without incurring unsustainable R&D costs. However, it is widely acknowledged that trends in industry R&D productivity have been moving in the opposite direction for a no. of years. Here, we present a detailed anal. based on comprehensive, recent, industry-wide data to identify the relative contributions of each of the steps in the drug discovery and development process to overall R&D productivity. We then propose specific strategies that could have the most substantial impact in improving R&D productivity.
- 10Bandyopadhyay, A.; Gao, J. Targeting Biomolecules with Reversible Covalent Chemistry. Curr. Opin. Chem. Biol. 2016, 34, 110– 116, DOI: 10.1016/j.cbpa.2016.08.011[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVOqtrrE&md5=1294e2befed2127ceec6a7e7ae0dd2f3Targeting biomolecules with reversible covalent chemistryBandyopadhyay, Anupam; Gao, JianminCurrent Opinion in Chemical Biology (2016), 34 (), 110-116CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)Interaction of biomols. typically proceeds in a highly selective and reversible manner, for which covalent bond formation has been largely avoided due to the potential difficulty of dissocn. However, employing reversible covalent warheads in drug design has given rise to covalent enzyme inhibitors that serve as powerful therapeutics, as well as mol. probes with exquisite target selectivity. This review article summarizes the recent advances in the development of reversible covalent chem. for biol. and medicinal applications. Specifically, we document the chem. strategies that allow for reversible modification of the three major classes of nucleophiles in biol.: thiols, alcs. and amines. Emphasis is given to the chem. mechanisms that underlie the development of these reversible covalent reactions and their utilization in biol.
- 11Bradshaw, J. M.; McFarland, J. M.; Paavilainen, V. O.; Bisconte, A.; Tam, D.; Phan, V. T.; Romanov, S.; Finkle, D.; Shu, J.; Patel, V.; Ton, T.; Li, X.; Loughhead, D. G.; Nunn, P. A.; Karr, D. E.; Gerritsen, M. E.; Funk, J. O.; Owens, T. D.; Verner, E.; Brameld, K. A.; Hill, R. J.; Goldstein, D. M.; Taunton, J. Prolonged and Tunable Residence Time Using Reversible Covalent Kinase Inhibitors. Nat. Chem. Biol. 2015, 11 (7), 525– 531, DOI: 10.1038/nchembio.1817[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFeju7%252FE&md5=85a73d9ecd62695d03166f7009c68dd8Prolonged and tunable residence time using reversible covalent kinase inhibitorsBradshaw, J. Michael; McFarland, Jesse M.; Paavilainen, Ville O.; Bisconte, Angelina; Tam, Danny; Phan, Vernon T.; Romanov, Sergei; Finkle, David; Shu, Jin; Patel, Vaishali; Ton, Tony; Li, Xiaoyan; Loughhead, David G.; Nunn, Philip A.; Karr, Dane E.; Gerritsen, Mary E.; Funk, Jens Oliver; Owens, Timothy D.; Verner, Erik; Brameld, Ken A.; Hill, Ronald J.; Goldstein, David M.; Taunton, JackNature Chemical Biology (2015), 11 (7), 525-531CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here the authors made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, the authors identified potent and selective BTK inhibitors that demonstrated biochem. residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.
- 12Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. J. Med. Chem. 2014, 57 (23), 10072– 10079, DOI: 10.1021/jm501412a[ACS Full Text], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVyjtLbM&md5=3ccf59ab7a494655185e5eb5becf8c48Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible InhibitorsFlanagan, Mark E.; Abramite, Joseph A.; Anderson, Dennis P.; Aulabaugh, Ann; Dahal, Upendra P.; Gilbert, Adam M.; Li, Chao; Montgomery, Justin; Oppenheimer, Stacey R.; Ryder, Tim; Schuff, Brandon P.; Uccello, Daniel P.; Walker, Gregory S.; Wu, Yan; Brown, Matthew F.; Chen, Jinshan M.; Hayward, Matthew M.; Noe, Mark C.; Obach, R. Scott; Philippe, Laurence; Shanmugasundaram, Veerabahu; Shapiro, Michael J.; Starr, Jeremy; Stroh, Justin; Che, YeJournal of Medicinal Chemistry (2014), 57 (23), 10072-10079CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry addnl. risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biol. relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.
- 13Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.; Horning, B. D.; González-Páez, G. E.; Chatterjee, S.; Lanning, B. R.; Teijaro, J. R.; Olson, A. J.; Wolan, D. W.; Cravatt, B. F. Proteome-Wide Covalent Ligand Discovery in Native Biological Systems. Nature 2016, 534 (7608), 570– 574, DOI: 10.1038/nature18002[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVSksbnN&md5=6fab087c042de735f8a5931e9f5b946cProteome-wide covalent ligand discovery in native biological systemsBackus, Keriann M.; Correia, Bruno E.; Lum, Kenneth M.; Forli, Stefano; Horning, Benjamin D.; Gonzalez-Paez, Gonzalo E.; Chatterjee, Sandip; Lanning, Bryan R.; Teijaro, John R.; Olson, Arthur J.; Wolan, Dennis W.; Cravatt, Benjamin F.Nature (London, United Kingdom) (2016), 534 (7608), 570-574CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Small mols. are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-mol. ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-mol. probes for proteins that have proven difficult to target using high-throughput screening of complex compd. libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-mol. probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quant. anal. of cysteine-reactive small-mol. fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chem. probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compds. that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines vs. primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compds. that can illuminate protein functions in native biol. systems.
- 14Miller, R. M.; Paavilainen, V. O.; Krishnan, S.; Serafimova, I. M.; Taunton, J. Electrophilic Fragment-Based Design of Reversible Covalent Kinase Inhibitors. J. Am. Chem. Soc. 2013, 135 (14), 5298– 5301, DOI: 10.1021/ja401221b[ACS Full Text], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltVClu7g%253D&md5=c6100b56ce6f587f6afb834aeafec351Electrophilic Fragment-Based Design of Reversible Covalent Kinase InhibitorsMiller, Rand M.; Paavilainen, Ville O.; Krishnan, Shyam; Serafimova, Iana M.; Taunton, JackJournal of the American Chemical Society (2013), 135 (14), 5298-5301CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Fragment-based ligand design and covalent targeting of noncatalytic cysteines have been employed to develop potent and selective kinase inhibitors. Here, we combine these approaches, starting with a panel of low-mol.-wt., heteroaryl-substituted cyanoacrylamides, which we have previously shown to form reversible covalent bonds with cysteine thiols. Using this strategy, we identify electrophilic fragments with sufficient ligand efficiency and selectivity to serve as starting points for the first reported inhibitors of the MSK1 C-terminal kinase domain. Guided by X-ray co-crystal structures, indazole fragment 1 was elaborated to afford 12 (RMM-46), a reversible covalent inhibitor that exhibits high ligand efficiency and selectivity for MSK/RSK-family kinases. At nanomolar concns., 12 blocked activation of cellular MSK and RSK, as well as downstream phosphorylation of the crit. transcription factor, CREB.
- 15Jöst, C.; Nitsche, C.; Scholz, T.; Roux, L.; Klein, C. D. Promiscuity and Selectivity in Covalent Enzyme Inhibition: A Systematic Study of Electrophilic Fragments. J. Med. Chem. 2014, 57 (18), 7590– 7599, DOI: 10.1021/jm5006918[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M%252FktlKisA%253D%253D&md5=d89c64d854194704eb068147961de4afPromiscuity and selectivity in covalent enzyme inhibition: a systematic study of electrophilic fragmentsJost Christian; Nitsche Christoph; Scholz Therese; Roux Lionel; Klein Christian DJournal of medicinal chemistry (2014), 57 (18), 7590-9 ISSN:.Covalent ligand-target interactions offer significant pharmacological advantages. However, off-target reactivity of the reactive groups, which usually have electrophilic properties, must be minimized, and the selectivity of irreversible inhibitors is a crucial requirement. We therefore performed a systematic study to determine the selectivity of several electrophilic groups that can be used as building blocks for covalently binding ligands. Six reactive groups with modulated electrophilicity were combined with 11 nonreactive moieties, resulting in a small combinatorial library of 72 fragment-like compounds. These compounds were screened against a group of 11 enzyme targets to assess their selectivity and their potential for promiscuous binding to proteins. The assay results showed a considerably lower degree of promiscuity than initially expected, even for those members of the screening collection that contain supposedly highly reactive electrophiles.
- 16Kathman, S. G.; Xu, Z.; Statsyuk, A. V. A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases. J. Med. Chem. 2014, 57 (11), 4969– 4974, DOI: 10.1021/jm500345q[ACS Full Text], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXovVWiur0%253D&md5=39dee964ac1ba26ed373eedbd86102acA Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine ProteasesKathman, Stefan G.; Xu, Ziyang; Statsyuk, Alexander V.Journal of Medicinal Chemistry (2014), 57 (11), 4969-4974CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A novel fragment-based drug discovery approach is reported which irreversibly tethers drug-like fragments to catalytic cysteines. Acrylamides, acylaminoacrylates, vinylsulfonamides, and acylaminopropenyl sulfones were prepd. and the dependence of their rates of reaction with N-acetylcysteine Me ester on their structure was detd.; of the compds. tested, the rate of Michael addn. of N-acetylcysteine Me ester to acylaminoacrylates depended least on the acyl moiety. A library of 100 fragment-substituted acylaminoacrylates was prepd.; addn. of subsets of the library to papain followed by mass spectrometric anal. identified three acylaminoacrylates which selectively reacted with the cysteine protease papain. The kinetics of the inhibition of papain by the acylaminoacrylates, the effect of known inhibitors of papain on its inhibition by the acylaminoacrylates, and the lack of inhibition of other cysteine proteases (human rhinovirus 3C protease, the catalytic domain of the deubiquitinase USP08, and the E2 ubiquitin-conjugating enzyme UbcH7) by the acylaminoacrylates supported their identification as selective and irreversible papain inhibitors.
- 17Ostrem, J. M.; Peters, U.; Sos, M. L.; Wells, J. A.; Shokat, K. M. K-Ras(G12C) Inhibitors Allosterically Control GTP Affinity and Effector Interactions. Nature 2013, 503 (7477), 548– 551, DOI: 10.1038/nature12796[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVGqs7jO&md5=27c1e7d8ef6b7e4b2919e9f00a073923K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactionsOstrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.Nature (London, United Kingdom) (2013), 503 (7477), 548-551CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally assocd. with poor response to std. therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concn. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small mols. that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compds. rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallog. studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favor GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.
- 18Zimmermann, G.; Rieder, U.; Bajic, D.; Vanetti, S.; Chaikuad, A.; Knapp, S.; Scheuermann, J.; Mattarella, M.; Neri, D. A Specific and Covalent JNK-1 Ligand Selected from an Encoded Self-Assembling Chemical Library. Chem. - Eur. J. 2017, 23 (34), 8152– 8155, DOI: 10.1002/chem.201701644[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXos1eitbY%253D&md5=5109df70628c1a2178cab73d645d82c9A Specific and Covalent JNK-1 Ligand Selected from an Encoded Self-Assembling Chemical LibraryZimmermann, Gunther; Rieder, Ulrike; Bajic, Davor; Vanetti, Sara; Chaikuad, Apirat; Knapp, Stefan; Scheuermann, Joerg; Mattarella, Martin; Neri, DarioChemistry - A European Journal (2017), 23 (34), 8152-8155CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)We describe the construction of a DNA-encoded chem. library comprising 148 135 members, generated through the self-assembly of two sub-libraries, contg. 265 and 559 members, resp. The library was designed to contain building blocks potentially capable of forming covalent interactions with target proteins. Selections performed with JNK1, a kinase contg. a conserved cysteine residue close to the ATP binding site, revealed the preferential enrichment of a 2-phenoxynicotinic acid moiety (building block A82) and a 4-(3,4-difluorophenyl)-4-oxobut-2-enoic acid moiety (building block B272). When the two compds. were joined by a short PEG linker, the resulting bidentate binder (A82-L-B272) was able to covalently modify JNK1 in the presence of a large molar excess of glutathione (0.5 mM), used to simulate intracellular reducing conditions. By contrast, derivs. of the individual building blocks were not able to covalently modify JNK1 in the same exptl. conditions. The A82-L-B272 ligand was selective over related kinases (BTK and GAK), which also contain targetable cysteine residues in the vicinity of the active site.
- 19Zambaldo, C.; Daguer, J.-P.; Saarbach, J.; Barluenga, S.; Winssinger, N. Screening for Covalent Inhibitors Using DNA-Display of Small Molecule Libraries Functionalized with Cysteine Reactive Moieties. MedChemComm 2016, 7 (7), 1340– 1351, DOI: 10.1039/C6MD00242K[Crossref], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVCis77I&md5=799993f7f231acd4a9951106d1f9de56Screening for covalent inhibitors using DNA-display of small molecule libraries functionalized with cysteine reactive moietiesZambaldo, C.; Daguer, J.-P.; Saarbach, J.; Barluenga, S.; Winssinger, N.MedChemComm (2016), 7 (7), 1340-1351CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)DNA-encoded chem. libraries are increasingly used to identify leads for drug discovery or chem. biol. Despite the resurging interest in covalent inhibitors, libraries are typically designed with synthon filtered out for reactive functionalities that can engage a target through covalent interactions. Herein, we report the synthesis of two libraries contg. Michael acceptors to identify cysteine reactive ligands. We developed a simple procedure to discriminate between covalent and high affinity non-covalent inhibitors using DNA display of the library in a microarray format. The methodol. was validated with known covalent and high affinity non-covalent kinase inhibitors. Screening of the library revealed novel covalent inhibitors for MEK2 and ERBB2.
- 20Strelow, J. M. A Perspective on the Kinetics of Covalent and Irreversible Inhibition. SLAS Discov. 2017, 22 (1), 3– 20, DOI: 10.1177/1087057116671509[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlSntrbJ&md5=bd404e6b26df20b8761cd38f0f3c3541A perspective on the kinetics of covalent and irreversible inhibitionStrelow, John M.SLAS Discovery (2017), 22 (1), 3-20CODEN: SDLIBT; ISSN:2472-5560. (Sage Publications)The clin. and com. success of covalent drugs has prompted a renewed and more deliberate pursuit of covalent and irreversible mechanisms within drug discovery. A covalent mechanism can produce potent inhibition in a biochem., cellular, or in vivo setting. In many cases, teams choose to focus on the consequences of the covalent event, defined by an IC50 value. In a biochem. assay, the IC50 may simply reflect the target protein concn. in the assay. What has received less attention is the importance of the rate of covalent modification, defined by kinact/KI. The kinact/KI is a rate const. describing the efficiency of covalent bond formation resulting from the potency (KI) of the first reversible binding event and the max. potential rate (kinact) of inactivation. In this perspective, it is proposed that the kinact/KI should be employed as a crit. parameter to identify covalent inhibitors, interpret structure-activity relationships (SARs), translate activity from biochem. assays to the cell, and more accurately define selectivity. It is also proposed that a physiol. relevant kinact/KI and an (unbound) AUC generated from a pharmacokinetic profile reflecting direct exposure of the inhibitor to the target protein are two crit. determinants of in vivo covalent occupancy. A simple equation is presented to define this relationship and improve the interpretation of covalent and irreversible kinetics.
- 21Miyahisa, I.; Sameshima, T.; Hixon, M. S. Rapid Determination of the Specificity Constant of Irreversible Inhibitors (Kinact/Ki) by Means of an Endpoint Competition Assay. Angew. Chem., Int. Ed. 2015, 54 (47), 14099– 14102, DOI: 10.1002/anie.201505800[Crossref], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsF2ktr3N&md5=9491844cd2fde8dda986189aff7800d8Rapid Determination of the Specificity Constant of Irreversible Inhibitors (kinact/KI) by Means of an Endpoint Competition AssayMiyahisa, Ikuo; Sameshima, Tomoya; Hixon, Mark S.Angewandte Chemie, International Edition (2015), 54 (47), 14099-14102CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Owing to their covalent target occupancy, irreversible inhibitors require low exposures and offer long duration, and their use thus represents a powerful strategy for achieving pharmacol. efficacy. Importantly, the potency metric of irreversible inhibitors is kinact/KI not IC50. A simple approach to measuring kinact/KI was developed that makes use of an irreversible probe for competitive assays run to completion against test compds. In this system, the kinact/KI value of the test compd. is equal to (kinact/KI)probe×[probe]/IC50. The advantages of this method include simplicity, high throughput, and application to all target classes, and it only requires an in-depth kinetic evaluation of the probe.
- 22Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry. Annu. Rev. Biochem. 2008, 77 (1), 383– 414, DOI: 10.1146/annurev.biochem.75.101304.124125[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXos1ekur0%253D&md5=2f2c471169d2473f7024eeb41d4eaecdActivity-based protein profiling: From enzyme chemistry to proteomic chemistryCravatt, Benjamin F.; Wright, Aaron T.; Kozarich, John W.Annual Review of Biochemistry (2008), 77 (), 383-414CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chem. proteomic strategy to characterize enzyme function directly in native biol. systems on a global scale. Here, the authors review the basic technol. of ABPP, the enzyme classes addressable by this method, and the biol. discoveries attributable to its application.
- 23Lanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.; Hett, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.; Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.; Whiteley, L. O.; Hayward, M. M.; Cravatt, B. F. A Road Map to Evaluate the Proteome-Wide Selectivity of Covalent Kinase Inhibitors. Nat. Chem. Biol. 2014, 10 (9), 760– 767, DOI: 10.1038/nchembio.1582[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFygs7nO&md5=6f17b0d1b00e088f37c511eea577d290A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitorsLanning, Bryan R.; Whitby, Landon R.; Dix, Melissa M.; Douhan, John; Gilbert, Adam M.; Hett, Erik C.; Johnson, Theodore O.; Joslyn, Chris; Kath, John C.; Niessen, Sherry; Roberts, Lee R.; Schnute, Mark E.; Wang, Chu; Hulce, Jonathan J.; Wei, Baoxian; Whiteley, Laurence O.; Hayward, Matthew M.; Cravatt, Benjamin F.Nature Chemical Biology (2014), 10 (9), 760-767CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quant. MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an exptl. road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.
- 24Zaro, B. W.; Whitby, L. R.; Lum, K. M.; Cravatt, B. F. Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors. J. Am. Chem. Soc. 2016, 138 (49), 15841– 15844, DOI: 10.1021/jacs.6b10589[ACS Full Text], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFyrtbbI&md5=830d336c87c6efa70659545c46c87b4eMetabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible InhibitorsZaro, Balyn W.; Whitby, Landon R.; Lum, Kenneth M.; Cravatt, Benjamin F.Journal of the American Chemical Society (2016), 138 (49), 15841-15844CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrophilic small mols. are an important class of chem. probes and drugs that produce biol. effects by irreversibly modifying proteins. Examples of electrophilic drugs include covalent kinase inhibitors that are used to treat cancer and the multiple sclerosis drug di-Me fumarate. Optimized covalent drugs typically inactivate their protein targets rapidly in cells, but ensuing time-dependent, off-target protein modification can erode selectivity and diminish the utility of reactive small mols. as chem. probes and therapeutics. Here, the authors describe an approach to confer kinetic selectivity to electrophilic drugs. The authors show that an analog of the covalent Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib bearing a fumarate ester electrophile is vulnerable to enzymic metab. on a time-scale that preserves rapid and sustained BTK inhibition, while thwarting more slowly accumulating off-target reactivity in cell and animal models. These findings demonstrate that metabolically labile electrophilic groups can endow covalent drugs with kinetic selectivity to enable perturbation of proteins and biochem. pathways with greater precision.
- 25Serafimova, I. M.; Pufall, M. A.; Krishnan, S.; Duda, K.; Cohen, M. S.; Maglathlin, R. L.; McFarland, J. M.; Miller, R. M.; Frödin, M.; Taunton, J. Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles. Nat. Chem. Biol. 2012, 8 (5), 471– 476, DOI: 10.1038/nchembio.925[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvVKitrw%253D&md5=6455a9a029714beb317286f71ac1326bReversible targeting of noncatalytic cysteines with chemically tuned electrophilesSerafimova, Iana M.; Pufall, Miles A.; Krishnan, Shyam; Duda, Katarzyna; Cohen, Michael S.; Maglathlin, Rebecca L.; McFarland, Jesse M.; Miller, Rand M.; Froedin, Morten; Taunton, JackNature Chemical Biology (2012), 8 (5), 471-476CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacol. potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissocg., covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chem.-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.
- 26Noe, M. C.; Gilbert, A. M. Targeted Covalent Enzyme Inhibitors. In Annual Reports in Medicinal Chemistry; Desai, M. C., Ed.; Academic Press, 2012; Vol. 47, pp. 413– 439. DOI: 10.1016/B978-0-12-396492-2.00027-8 .
- 27Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing Irreversible Inhibitors of the Protein Kinase Cysteinome. Chem. Biol. 2013, 20 (2), 146– 159, DOI: 10.1016/j.chembiol.2012.12.006[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtFWitbs%253D&md5=cef80823123bae88362e816a0a638133Developing Irreversible Inhibitors of the Protein Kinase CysteinomeLiu, Qingsong; Sabnis, Yogesh; Zhao, Zheng; Zhang, Tinghu; Buhrlage, Sara J.; Jones, Lyn H.; Gray, Nathanael S.Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 146-159CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)A review. Protein kinases are a large family of approx. 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues, such as tyrosine, serine, and threonine, that serves as a ubiquitous mechanism for cellular signal transduction. The clin. success of a no. of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large no. of kinases may represent therapeutically relevant targets. To date, the majority of clin. and preclin. kinase inhibitors are ATP competitive, noncovalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently, there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a no. of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency, and ability to validate pharmacol. specificity through mutation of the reactive cysteine residue. Here, we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biol. activity profiles. In addn., we provided an informatics assessment of the potential "kinase cysteinome" and discuss strategies for the efficient development of new covalent inhibitors.
- 28Miller, R. M.; Taunton, J. Targeting Protein Kinases with Selective and Semipromiscuous Covalent Inhibitors. In Methods in Enzymology; Shokat, K. M., Ed.; Academic Press, 2014; Vol. 548, pp 93– 116. DOI: 10.1016/B978-0-12-397918-6.00004-5 .
- 29Gilbert, A. M. Recent Advances in Irreversible Kinase Inhibitors. Pharm. Pat. Anal. 2014, 3 (4), 375– 386, DOI: 10.4155/ppa.14.24[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslSkurnN&md5=e0d3394e575a3fdb4e16d4627b83baa5Recent advances in irreversible kinase inhibitorsGilbert, Adam M.Pharmaceutical Patent Analyst (2014), 3 (4), 375-386CODEN: PPAHCN; ISSN:2046-8954. (Future Science Ltd.)Despite concerns of off-target selectivity and cytotoxicity, there has been a resurgence in interest in irreversible kinase inhibitors resulting in more than 60 disclosed patent and patent applications over the past 4 years. Many of these inhibitors possess several key advantages over their reversible counterparts. The patent literature from 2010 to 2013 has been reviewed and novel irreversible kinase inhibitors for Bruton's tyrosine kinase, epidermal growth factor receptor, Janus kinase 3, phosphoinsitide 3 and other kinases are disclosed and discussed. These inhibitors offer novel treatments for mantle cell lymphoma, non-small-cell lung cancer, autoimmune disorders and severe metastatic cancers. A future perspective is presented on the likelihood of clin. success of these agents as well as the potential for new uses of irreversible kinase inhibitors in the future.
- 30Adeniyi, A. A.; Muthusamy, R.; Soliman, M. E. New Drug Design with Covalent Modifiers. Expert Opin. Drug Discovery 2016, 11 (1), 79– 90, DOI: 10.1517/17460441.2016.1115478[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht12qtL8%253D&md5=9307f11db2da4d9853b49a97cec7e0e2New drug design with covalent modifiersAdeniyi, Adebayo A.; Muthusamy, Ramesh; Soliman, Mahmoud ESExpert Opinion on Drug Discovery (2016), 11 (1), 79-90CODEN: EODDBX; ISSN:1746-0441. (Taylor & Francis Ltd.)Introduction: A major challenge for drug design is the alarming increase in drug resistance, mutations and toxicity. In recent years, covalent drugs have become a promising option to address these problems, due to their significant advantages. These advantages include their ability to target rare, non-conserved residues, shallow binding sites of target proteins and their ability to retain their binding with a receptor for a very long time. Areas covered: This review shows the increasing progress in rational design and virtual screening of covalent drugs and the promising future of accurately predicting effective covalent drugs through in silico screening. Expert opinion: All the current clin. approved covalent drugs were discovered by chance instead of systematic design. There is a promising and commendable effort towards high-throughput screening and the accurate discovery of new covalent inhibitors, which may address the problems of drug resistance and mutation. However, despite the current progression, there is still a need for more rational attention to improve the covalent warhead for improved receptor interaction and selectivity.
- 31Baillie, T. A. Targeted Covalent Inhibitors for Drug Design. Angew. Chem., Int. Ed. 2016, 55 (43), 13408– 13421, DOI: 10.1002/anie.201601091[Crossref], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlOrtb%252FN&md5=18bed2ae08dcb4245a42b3070abc6354Targeted Covalent Inhibitors for Drug DesignBaillie, Thomas A.Angewandte Chemie, International Edition (2016), 55 (43), 13408-13421CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)In contrast to the traditional mechanism of drug action that relies on the reversible, noncovalent interaction of a ligand with its biol. target, a targeted covalent inhibitor (TCI) is designed such that the initial, reversible assocn. is followed by the formation of a covalent bond between an electrophile on the ligand and a nucleophilic center in the protein. Although this approach offers a variety of potential benefits (high potency and extended duration of action), concerns over the possible toxicol. consequences of protein haptenization have hindered the development of the TCI concept. Recently, approaches to mitigate the risk of serious adverse reactions to this new class of agent have emerged, thus stimulating interest in the field and leading to authorization of the first cadre of TCIs to be marketed. The covalent inhibitor approach is rapidly gaining acceptance as a valuable tool in drug discovery, and is poised to make a major impact on the design of enzyme inhibitors and receptor modulators.
- 32Hallenbeck, K. K.; Turner, D. M.; Renslo, A. R.; Arkin, M. R. Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery. Curr. Top. Med. Chem. 2016, 17, 4– 15, DOI: 10.2174/1568026616666160719163839
- 33Lagoutte, R.; Patouret, R.; Winssinger, N. Covalent Inhibitors: An Opportunity for Rational Target Selectivity. Curr. Opin. Chem. Biol. 2017, 39, 54– 63, DOI: 10.1016/j.cbpa.2017.05.008[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpslGmsb4%253D&md5=2f7fac86ca6f6963050e1d6a69ef453eCovalent inhibitors: an opportunity for rational target selectivityLagoutte, Roman; Patouret, Remi; Winssinger, NicolasCurrent Opinion in Chemical Biology (2017), 39 (), 54-63CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)There is a resurging interest in compds. that engage their target through covalent interactions. Cysteine's thiol is endowed with enhanced reactivity, making it the nucleophile of choice for covalent engagement with a ligand aligning an electrophilic trap with a cysteine residue in a target of interest. The paucity of cysteine in the proteome coupled to the fact that closely related proteins do not necessarily share a given cysteine residue enable a level of unprecedented rational target selectivity. The recent demonstration that a lysine's amine can also be engaged covalently with a mild electrophile extends the potential of covalent inhibitors. The growing database of protein structures facilitates the discovery of covalent inhibitors while the advent of proteomic technologies enables a finer resoln. in the selectivity of covalently engaged proteins. Here, we discuss recent examples of discovery and design of covalent inhibitors.
- 34De Cesco, S.; Kurian, J.; Dufresne, C.; Mittermaier, A. K.; Moitessier, N. Covalent Inhibitors Design and Discovery. Eur. J. Med. Chem. 2017, 138, 96– 114, DOI: 10.1016/j.ejmech.2017.06.019[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKhsLrI&md5=551c9a9c4bd69d4ad4d9156c315d5a94Covalent inhibitors design and discoveryDe Cesco, Stephane; Kurian, Jerry; Dufresne, Caroline; Mittermaier, Anthony K.; Moitessier, NicolasEuropean Journal of Medicinal Chemistry (2017), 138 (), 96-114CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)In the history of therapeutics, covalent drugs occupy a very distinct category. While representing a significant fraction of the drugs on the market, very few have been deliberately designed to interact covalently with their biol. target. In this review, the prevalence of covalent drugs will first be briefly covered, followed by an introduction to their mechanisms of action and more detailed discussions of their discovery and the development of safe and efficient covalent enzyme inhibitors. All stages of a drug discovery program will be covered, from target considerations to lead optimization, strategies to tune reactivity and computational methods. The goal of this article is to provide an overview of the field and to outline good practices that are needed for the proper assessment and development of covalent inhibitors as well as a good understanding of the potential and limitations of current computational methods for the design of covalent drugs.
- 35Chaikuad, A.; Koch, P.; Laufer, S. A.; Knapp, S. The Cysteinome of Protein Kinases as a Target in Drug Development. Angew. Chem., Int. Ed. 2018, 57 (16), 4372– 4385, DOI: 10.1002/anie.201707875[Crossref], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFGgs7c%253D&md5=311ba5ab53dc4a5dc1dc92ddd4b6d4c2The Cysteinome of Protein Kinases as a Target in Drug DevelopmentChaikuad, Apirat; Koch, Pierre; Laufer, Stefan A.; Knapp, StefanAngewandte Chemie, International Edition (2018), 57 (16), 4372-4385CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Drugs that function through covalent bond formation represent a considerable fraction of the authors' repository of effective medicines but safety concerns and the complexity of developing covalent inhibitors has rendered covalent targeting a less attractive strategy for rational drug design. The recent approval of four covalent kinase inhibitors and the development of highly potent covalent kinase probes with exceptional selectivity has raised significant interest in industry and academic research and validated the concept of covalent kinase targeting for clin. applications. The abundance of cysteines at diverse positions in and around the kinase active site suggests that a large fraction of kinases can be targeted by covalent inhibitors. Herein, the authors review recent developments of this rapidly growing area in kinase drug development and highlight the unique opportunities and challenges of this strategy.
- 36Zhao, Z.; Bourne, P. E. Progress with Covalent Small-Molecule Kinase Inhibitors. Drug Discovery Today 2018, 23 (3), 727– 735, DOI: 10.1016/j.drudis.2018.01.035[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslShurg%253D&md5=f97a19f53a72044812a778ee9351fe94Progress with covalent small-molecule kinase inhibitorsZhao, Zheng; Bourne, Philip E.Drug Discovery Today (2018), 23 (3), 727-735CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)With reduced risk of toxicity and high selectivity, covalent small-mol. kinase inhibitors (CSKIs) have emerged rapidly. Through the lens of structural system pharmacol., here we review this rapid progress by considering design strategies and the challenges and opportunities offered by current CSKIs.
- 37Lonsdale, R.; Ward, R. A. Structure-Based Design of Targeted Covalent Inhibitors. Chem. Soc. Rev. 2018, 47 (11), 3816– 3830, DOI: 10.1039/C7CS00220C[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVGiurs%253D&md5=a7e31b23cf8ecfcad1397ad10ea10b7bStructure-based design of targeted covalent inhibitorsLonsdale, Richard; Ward, Richard A.Chemical Society Reviews (2018), 47 (11), 3816-3830CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Covalent inhibition is a rapidly growing discipline within drug discovery. Many historical covalent inhibitors were discovered by serendipity, with such a mechanism of action often regarded as undesirable due to potential toxicity issues. Recent progress has seen a major shift in this outlook, as covalent inhibition shows promise for targets where previous efforts to identify non-covalent small mol. inhibitors have failed. Targeted covalent inhibitors (TCIs) can offer drug discovery scientists the ability to increase the potency and/or selectivity of small mol. inhibitors, by attachment of reactive functional groups designed to covalently bind to specific sites in a target. In this tutorial review we introduce the broader concept of covalent inhibition, discuss the potential benefits and challenges of such an approach, and provide an overview of the current status of the field. We also describe some strategies and computational tools to enable successful covalent drug discovery.
- 38Ferguson, F. M.; Gray, N. S. Kinase Inhibitors: The Road Ahead. Nat. Rev. Drug Discovery 2018, 17 (5), 353– 377, DOI: 10.1038/nrd.2018.21[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXks12ktbg%253D&md5=34e42911d8a70301905460c3507e737aKinase inhibitors: the road aheadFerguson, Fleur M.; Gray, Nathanael S.Nature Reviews Drug Discovery (2018), 17 (5), 353-377CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review. Receptor tyrosine kinase signaling pathways have been successfully targeted to inhibit proliferation and angiogenesis for cancer therapy. However, kinase deregulation has been firmly demonstrated to play an essential role in virtually all major disease areas. Kinase inhibitor drug discovery programs have recently broadened their focus to include an expanded range of kinase targets and therapeutic areas. In this Review, we provide an overview of the novel targets, biol. processes and disease areas that kinase-targeting small mols. are being developed against, highlight the assocd. challenges and assess the strategies and technologies that are enabling efficient generation of highly optimized kinase inhibitors.
- 39Pettinger, J.; Jones, K.; Cheeseman, M. D. Lysine-Targeting Covalent Inhibitors. Angew. Chem., Int. Ed. 2017, 56 (48), 15200– 15209, DOI: 10.1002/anie.201707630[Crossref], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslejtr3P&md5=4a6bb561e53c165fa3c7c85953bb4736Lysine-Targeting Covalent InhibitorsPettinger, Jonathan; Jones, Keith; Cheeseman, Matthew D.Angewandte Chemie, International Edition (2017), 56 (48), 15200-15209CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Targeted covalent inhibitors have gained widespread attention in drug discovery as a validated method to circumvent acquired resistance in oncol. This strategy exploits small-mol./protein crystal structures to design tightly binding ligands with appropriately positioned electrophilic warheads. While most focus has been on targeting binding-site cysteine residues, targeting nucleophilic lysine residues can also represent a viable approach to irreversible inhibition. However, owing to the basicity of the ε-amino group in lysine, this strategy generates a no. of specific challenges. Herein, the authors review the key principles for inhibitor design, give historical examples, and present recent developments that demonstrate the potential of lysine targeting for future drug discovery.
- 40Jackson, P. A.; Widen, J. C.; Harki, D. A.; Brummond, K. M. Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions. J. Med. Chem. 2017, 60 (3), 839– 885, DOI: 10.1021/acs.jmedchem.6b00788[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitV2rtbnM&md5=36d3be5bf688c4e4a8cf4bcf42073009Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition ReactionsJackson, Paul A.; Widen, John C.; Harki, Daniel A.; Brummond, Kay M.Journal of Medicinal Chemistry (2017), 60 (3), 839-885CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Although Michael acceptors display a potent and broad spectrum of bioactivity, they have largely been ignored in drug discovery because of their presumed indiscriminate reactivity. As such, a dearth of information exists relevant to the thiol reactivity of natural products and their analogs possessing this moiety. In the midst of recently approved acrylamide-contg. drugs, it is clear that a good understanding of the hetero-Michael addn. reaction and the relative reactivities of biol. thiols with Michael acceptors under physiol. conditions is needed for the design and use of these compds. as biol. tools and potential therapeutics. This Perspective provides information that will contribute to this understanding, such as kinetics of thiol addn. reactions, bioactivities, as well as steric and electronic factors that influence the electrophilicity and reversibility of Michael acceptors. This Perspective is focused on α,β-unsatd. carbonyls given their preponderance in bioactive natural products.
- 41Baslé, E.; Joubert, N.; Pucheault, M. Protein Chemical Modification on Endogenous Amino Acids. Chem. Biol. 2010, 17 (3), 213– 227, DOI: 10.1016/j.chembiol.2010.02.008[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjvFKhurY%253D&md5=82a023a2f4e96136205bdb19901bd401Protein Chemical Modification on Endogenous Amino AcidsBasle, Emmanuel; Joubert, Nicolas; Pucheault, MathieuChemistry & Biology (Cambridge, MA, United States) (2010), 17 (3), 213-227CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)A review. Chem. modification of protein is an arduous but fruitful task. Many chem. methods have been developed for such purpose by carefully balancing reactivity and selectivity. Now both chemists and biologists have in hand an arsenal of tools from which they can select a relevant reaction to tackle their problems. This review focuses on the various chem. transformations available for selective modification of proteins. It also provides a brief overview of some of their main applications, including detection of protein interactions, prepn. of bioconjugates, and protein microarrays.
- 42Boutureira, O.; Bernardes, G. J. L. Advances in Chemical Protein Modification. Chem. Rev. 2015, 115 (5), 2174– 2195, DOI: 10.1021/cr500399p[ACS Full Text], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtV2nu7k%253D&md5=d21a5407a59a7e12346c16e5db75ab91Advances in Chemical Protein ModificationBoutureira, Omar; Bernardes, Goncalo J. L.Chemical Reviews (Washington, DC, United States) (2015), 115 (5), 2174-2195CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Transition metal-free and -mediated approaches are covered.
- 43Shannon, D. A.; Weerapana, E. Covalent Protein Modification: The Current Landscape of Residue-Specific Electrophiles. Curr. Opin. Chem. Biol. 2015, 24, 18– 26, DOI: 10.1016/j.cbpa.2014.10.021[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFSlsrzF&md5=ef6f0fe9ed08ee0653fe0f19f87bdafcCovalent protein modification: the current landscape of residue-specific electrophilesShannon, D. Alexander; Weerapana, EranthieCurrent Opinion in Chemical Biology (2015), 24 (), 18-26CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Functional amino acids that play crit. roles in catalysis and regulation are known to display elevated nucleophilicity and can be selectively targeted for covalent modification by reactive electrophiles. Chem.-proteomic platforms, such as activity-based protein profiling (ABPP), exploit this reactivity by utilizing chem. probes to covalently modify active-site residues to inform on the functional state of enzymes within complex proteomes. These and other applications rely on the availability of a diverse array of electrophiles and detailed knowledge of the reactivity and amino-acid selectivity of these groups. Here, we survey the current landscape of electrophiles that covalently target various nucleophilic amino acids in proteins and highlight proteomic applications that have benefited from the unique properties of these electrophiles.
- 44Gunnoo, S. B.; Madder, A. Chemical Protein Modification through Cysteine. ChemBioChem 2016, 17 (7), 529– 553, DOI: 10.1002/cbic.201500667[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjvVyrsrY%253D&md5=275b9e4e5770811079411cb048f47427Chemical Protein Modification through CysteineGunnoo, Smita B.; Madder, AnnemiekeChemBioChem (2016), 17 (7), 529-553CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chem. modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance-both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chem. protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.
- 45Dondoni, A.; Marra, A. SuFEx: A Metal-Free Click Ligation for Multivalent Biomolecules. Org. Biomol. Chem. 2017, 15 (7), 1549– 1553, DOI: 10.1039/C6OB02458K[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFGgurY%253D&md5=fcad56172f56f0d9bc17146097479495SuFEx: a metal-free click ligation for multivalent biomoleculesDondoni, Alessandro; Marra, AlbertoOrganic & Biomolecular Chemistry (2017), 15 (7), 1549-1553CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)A review. The Sulfur(VI) Fluoride Exchange (SuFEx), revived by Sharpless and co-workers from an unrecognized state, is an emerging new click reaction that is based on the high reactivity of sulfonyl fluorides and fluorosulfates with suitable nucleophiles such as silyl ethers and amines. Hence, we comment in this Perspective on the use of SuFEx for the synthesis of a new family of sugar contg. sulfonamides from the reaction of a glycosylsulfonyl fluoride with aliph. amines. We also highlight the applications of SuFEx to multivalent scaffolds such as tetraamino- and tetrafluorosulfonyl-calixarene leading to sulfonamide-linked sugar and iminosugar clusters. Finally, we report on the chemoselective functionalization of bovine serum albumin (BSA) which, owing to the amino group of lysine moieties, reacted with SO2F2 to give a multivalent BSA-SO2F system. The PEGylation of the same protein by coupling with a PEG-fluorosulfate is described as well.
- 46deGruyter, J. N.; Malins, L. R.; Baran, P. S. Residue-Specific Peptide Modification: A Chemist’s Guide. Biochemistry 2017, 56 (30), 3863– 3873, DOI: 10.1021/acs.biochem.7b00536[ACS Full Text], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKisLfK&md5=cb0a794b5077461762fd9fa43f4f39c2Residue-Specific Peptide Modification: A Chemist's GuidedeGruyter, Justine N.; Malins, Lara R.; Baran, Phil S.Biochemistry (2017), 56 (30), 3863-3873CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. Advances in bioconjugation and native protein modification are appearing at a blistering pace, making it increasingly time consuming for practitioners to identify the best chem. method to modify a specific amino acid residue in a complex setting. The purpose of this perspective is to provide an informative, graphically rich manual highlighting significant advances in the field over the past decade. This guide will help triage candidate methods for peptide alteration, and will serve as a starting point for those seeking to solve longstanding challenges.
- 47Hoch, D. G.; Abegg, D.; Adibekian, A. Cysteine-Reactive Probes and Their Use in Chemical Proteomics. Chem. Commun. 2018, 54 (36), 4501– 4512, DOI: 10.1039/C8CC01485J[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnsVegs7k%253D&md5=d91bb3d96048cb6c9516d69033b2365fCysteine-reactive probes and their use in chemical proteomicsHoch, Dominic G.; Abegg, Daniel; Adibekian, AlexanderChemical Communications (Cambridge, United Kingdom) (2018), 54 (36), 4501-4512CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. Proteomic profiling using bioorthogonal chem. probes that selectively react with certain amino acids is now a widely used method in life sciences to investigate enzymic activities, study posttranslational modifications and discover novel covalent inhibitors. Over the past two decades, researchers have developed selective probes for several different amino acids, including lysine, serine, cysteine, threonine, tyrosine, aspartate and glutamate. Among these amino acids, cysteines are particularly interesting due to their highly diverse and complex biochem. role in our cells. In this feature article, we focus on the chem. probes and methods used to study cysteines in complex proteomes.
- 48Cromm, P. M.; Crews, C. M. The Proteasome in Modern Drug Discovery: Second Life of a Highly Valuable Drug Target. ACS Cent. Sci. 2017, 3 (8), 830– 838, DOI: 10.1021/acscentsci.7b00252[ACS Full Text], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1Orsb3M&md5=91f26fd1133db650c0b4b4f43a461354The Proteasome in Modern Drug Discovery: Second Life of a Highly Valuable Drug TargetCromm, Philipp M.; Crews, Craig M.ACS Central Science (2017), 3 (8), 830-838CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)A review. As the central figure of the cellular protein degrdn. machinery, the proteasome is crit. for cell survival. Having been extensively targeted for inhibition, the constitutive proteasome has proven its role as a highly valuable drug target. However, recent advances in the protein homeostasis field suggest that addnl. chapters can be added to this successful story. For example, selective immunoproteasome inhibition promises high clin. efficacy for autoimmune disorders and inflammation, and proteasome inhibitors might serve as novel therapeutics for malaria or other microorganisms. Furthermore, utilizing the destructive force of the proteasome for selective degrdn. of essential drivers of human disorders has opened up a new and exciting area of drug discovery. Thus, the field of proteasome drug discovery still holds exciting questions to be answered and does not simply end with inhibiting the constitutive proteasome.
- 49Casimiro-Garcia, A.; Trujillo, J. I.; Vajdos, F.; Juba, B.; Banker, M. E.; Aulabaugh, A.; Balbo, P.; Bauman, J.; Chrencik, J.; Coe, J. W.; Czerwinski, R.; Dowty, M.; Knafels, J. D.; Kwon, S.; Leung, L.; Liang, S.; Robinson, R. P.; Telliez, J.-B.; Unwalla, R.; Yang, X.; Thorarensen, A. Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors. J. Med. Chem. 2018, 61, 10665– 10699, DOI: 10.1021/acs.jmedchem.8b01308[ACS Full Text], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitF2qtrjK&md5=12832ed66b8fc89d5f6ef9a3aa4ea20fIdentification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent InhibitorsCasimiro-Garcia, Agustin; Trujillo, John I.; Vajdos, Felix; Juba, Brian; Banker, Mary Ellen; Aulabaugh, Ann; Balbo, Paul; Bauman, Jonathan; Chrencik, Jill; Coe, Jotham W.; Czerwinski, Robert; Dowty, Martin; Knafels, John D.; Kwon, Soojin; Leung, Louis; Liang, Sidney; Robinson, Ralph P.; Telliez, Jean-Baptiste; Unwalla, Ray; Yang, Xin; Thorarensen, AtliJournal of Medicinal Chemistry (2018), 61 (23), 10665-10699CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Ongoing interest in the discovery of selective JAK3 inhibitors led us to design novel covalent inhibitors that engage the JAK3 residue Cys909 by cyanamide, a structurally and mechanistically differentiated electrophile from other cysteine reacting groups previously incorporated in JAK3 covalent inhibitors. Through crystallog., kinetic, and computational studies, interaction of cyanamide I with Cys909 was optimized leading to potent and selective JAK3 inhibitors as exemplified by II. In relevant cell-based assays and in agreement with previous results from this group, II demonstrated that selective inhibition of JAK3 is sufficient to drive JAK1/JAK3-mediated cellular responses. The contribution from extrahepatic processes to the clearance of cyanamide-based covalent inhibitors was also characterized using metabolic and pharmacokinetic data for I. This work also gave key insights into a productive approach to decrease glutathione/glutathione S-transferase-mediated clearance, a challenge typically encountered during the discovery of covalent kinase inhibitors.
- 50Li, T.; Maltais, R.; Poirier, D.; Lin, S.-X. Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide. J. Phys. Chem. Lett. 2018, 9 (18), 5275– 5280, DOI: 10.1021/acs.jpclett.8b02225[ACS Full Text], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1SitbbJ&md5=46fc3f11f2484b972c5b6ce626136e14Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl HalideLi, Tang; Maltais, Rene; Poirier, Donald; Lin, Sheng-XiangJournal of Physical Chemistry Letters (2018), 9 (18), 5275-5280CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) plays a pivotal role in the progression of estrogen-related diseases because of its involvement in the biosynthesis of estradiol (E2), constituting a valuable therapeutic target for endocrine treatment. In the present study, we successfully cocrystd. the enzyme with the reversible inhibitor 2-methoxy-16β-(m-carbamoylbenzyl)-E2 (2-MeO-CC-156) as well as the enzyme with the irreversible inhibitor 3-(2-bromoethyl)-16β-(m-carbamoylbenzyl)-17β-hydroxy-1,3,5(10)-estratriene (PBRM). The structures of ternary complexes of 17β-HSD1-2-MeO-CC-156-NADP+ and 17β-HSD1-PBRM-NADP+ comparatively show the formation of a covalent bond between His221 and the bromoethyl side chain of the inhibitor in the PBRM structure. A dynamic process including beneficial mol. interactions that favor the specific binding of a low-reactivity inhibitor and subsequent N-alkylation event through the participation of His221 in the enzyme catalytic site clearly demonstrates the covalent bond formation. This finding opens the door to a new design of alkyl halide-based specific covalent inhibitors as potential therapeutic agents for different enzymes, contributing to the development of highly efficient inhibitors.
- 51Kharenko, O. A.; Patel, R. G.; Brown, S. D.; Calosing, C.; White, A.; Lakshminarasimhan, D.; Suto, R. K.; Duffy, B. C.; Kitchen, D. B.; McLure, K. G.; Hansen, H. C.; van der Horst, E. H.; Young, P. R. Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine. J. Med. Chem. 2018, 61 (18), 8202– 8211, DOI: 10.1021/acs.jmedchem.8b00666[ACS Full Text], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1ensLjO&md5=1c3ae393a5f8a8c12ffb16e6f3798118Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a MethionineKharenko, Olesya A.; Patel, Reena G.; Brown, S. David; Calosing, Cyrus; White, Andre; Lakshminarasimhan, Damodharan; Suto, Robert K.; Duffy, Bryan C.; Kitchen, Douglas B.; McLure, Kevin G.; Hansen, Henrik C.; van der Horst, Edward H.; Young, Peter R.Journal of Medicinal Chemistry (2018), 61 (18), 8202-8211CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small mol. bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacol. Here the authors describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an x-ray crystal structure, the authors demonstrate that these inhibitors selectively form a covalent bond with Met 149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.
- 52Pearson, R. G.; Sobel, H. R.; Songstad, J. Nucleophilic Reactivity Constants toward Methyl Iodide and Trans-Dichlorodi(Pyridine)Platinum(II). J. Am. Chem. Soc. 1968, 90 (2), 319– 326, DOI: 10.1021/ja01004a021[ACS Full Text], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF1cXhtFOntbk%253D&md5=b118d94887322fd7cdda64874f873aadNucleophilic reactivity constants toward methyl iodide and trans-dichlorodi(pyridine)platinum(II)Pearson, Ralph G.; Sobel, Harold R.; Songstad, JonJournal of the American Chemical Society (1968), 90 (2), 319-26CODEN: JACSAT; ISSN:0002-7863.The rates of reaction of a no. of nucleophiles with MeI and trans-[Pt(py)2Cl2] have been measured in MeOH at 25°. Relative nucleophilic reactivity parameters, nMeI and nPt, have been calcd. It was not possible to correlate these nos. with each other or with other extra-kinetic data. Equations in the literature for predicting nucleophilic reactivity have only a limited range of usefulness. 32 references.
- 53Sardi, F.; Manta, B.; Portillo-Ledesma, S.; Knoops, B.; Comini, M. A.; Ferrer-Sueta, G. Determination of Acidity and Nucleophilicity in Thiols by Reaction with Monobromobimane and Fluorescence Detection. Anal. Biochem. 2013, 435 (1), 74– 82, DOI: 10.1016/j.ab.2012.12.017[Crossref], [PubMed], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXisVGlsbk%253D&md5=72eb72b04f8626d6cd592ddeb60f779dDetermination of acidity and nucleophilicity in thiols by reaction with monobromobimane and fluorescence detectionSardi, Florencia; Manta, Bruno; Portillo-Ledesma, Stephanie; Knoops, Bernard; Comini, Marcelo A.; Ferrer-Sueta, GerardoAnalytical Biochemistry (2013), 435 (1), 74-82CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)A method based on the differential reactivity of thiol and thiolate with monobromobimane (mBBr) has been developed to measure nucleophilicity and acidity of protein and low-mol.-wt. thiols. Nucleophilicity of the thiolate is measured as the pH-independent second-order rate const. of its reaction with mBBr. The ionization consts. of the thiols are obtained through the pH dependence of either second-order rate const. or initial rate of reaction. For readily available thiols, the apparent second-order rate const. is measured at different pHs and then plotted and fitted to an appropriate pH function describing the obsd. no. of ionization equil. For less available thiols, such as protein thiols, the initial rate of reaction is detd. in a wide range of pHs and fitted to the appropriate pH function. The method presented here shows excellent sensitivity, allowing the use of nanomolar concns. of reagents. The method is suitable for scaling and high-throughput screening. Example detns. of nucleophilicity and pKa are presented for captopril and cysteine as low-mol.-wt. thiols and for human peroxiredoxin 5 and Trypanosoma brucei monothiol glutaredoxin 1 as protein thiols.
- 54Pearson, R. G. Hard and Soft Acids and Bases. J. Am. Chem. Soc. 1963, 85 (22), 3533– 3539, DOI: 10.1021/ja00905a001[ACS Full Text], [CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2cXksV0%253D&md5=2333a18c8168b373b612f5f45e8f4b4cHard and soft acids and basesPearson, Ralph G.Journal of the American Chemical Society (1963), 85 (22), 3533-9CODEN: JACSAT; ISSN:0002-7863.A number of Lewis acids of diverse types are classified as (a) or (b) following the criterion of Ahrland, et al. (CA 53, 960c). Other, auxiliary criteria are proposed. Class (a) acids prefer to bind to "hard" or nonpolarizable bases. Class (b) acids prefer to bind to "soft" or polarizable bases. Since class (a) acids are themselves "hard" and since class (b) acids are "soft" a simple, useful rule is proposed: hard acids bind strongly to hard bases and soft acids bind strongly to soft bases. The explanations for such behavior include: (1) various degrees of ionic and covalent σ-bonding; (2) π-bonding; (3) electron correlation phenomena; (4) solvation effects.
- 55Awoonor-Williams, E.; Rowley, C. N. Evaluation of Methods for the Calculation of the PKa of Cysteine Residues in Proteins. J. Chem. Theory Comput. 2016, 12 (9), 4662– 4673, DOI: 10.1021/acs.jctc.6b00631[ACS Full Text], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlOqtbvM&md5=f2d43fd808bdfb63fe2b19639ab4e64cEvaluation of Methods for the Calculation of the pKa of Cysteine Residues in ProteinsAwoonor-Williams, Ernest; Rowley, Christopher N.Journal of Chemical Theory and Computation (2016), 12 (9), 4662-4673CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)Methods for the calcn. of the pK of ionizable amino acids are valuable tools for understanding pH-dependent properties of proteins. Cysteine is unique among the amino acids because of the chem. reactivity of its thiol group (S-H), which plays an instrumental role in several biochem. and regulatory functions. The acidity of noncatalytic cysteine residues is a factor in their susceptibility to chem. modification. Despite the plethora of existing pKa computing methods, no definitive protocol exists for accurately calcg. the pKa's of cysteine residues in proteins. A cysteine pKa test set was developed, which is comprised of 18 cysteine residues in 12 proteins where the pKa's have been detd. exptl. and an exptl. structure is available. The pKa's of these residues were calcd. using three methods that use an implicit solvent model (H++, MCCE, and PROPKA) and an all-atom replica exchange thermodn. integration approach with the CHARMM36 and AMBER ff99SB-ILDNP force fields. The models that use implicit solvation methods were generally unreliable in predicting cysteine residue pKa's, with RMSDs between 3.41 and 4.72 pKa units. On av., the explicit solvent methods performed better than the implicit solvent methods. RMSD values of 2.40 and 3.20 were obtained for simulations with the CHARMM36 and AMBER ff99SB-ILDNP force fields, resp. Further development of these methods is necessary because the performance of the best method is similar to that of the null-model (RMSD = 2.74) and these differences in RMSD are of limited statistical significance given the small size of the authors' test set.
- 56Grimsley, G. R.; Scholtz, J. M.; Pace, C. N. A Summary of the Measured PK Values of the Ionizable Groups in Folded Proteins. Protein Sci. 2008, 18, 247– 251, DOI: 10.1002/pro.19
- 57Alcock, L. J.; Perkins, M. V.; Chalker, J. M. Chemical Methods for Mapping Cysteine Oxidation. Chem. Soc. Rev. 2018, 47 (1), 231– 268, DOI: 10.1039/C7CS00607A[Crossref], [PubMed], [CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFyisbvE&md5=498906bd9e79704dfd5ffd78791d25caChemical methods for mapping cysteine oxidationAlcock, Lisa J.; Perkins, Michael V.; Chalker, Justin M.Chemical Society Reviews (2018), 47 (1), 231-268CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Cysteine residues in proteins are subject to diverse redox chem. Oxidn. of cysteine to S-nitrosocysteine, cysteine sulfenic and sulfinic acids, disulfides and persulfides are a few prominent examples of these oxidative post-translational modifications. In living organisms, these modifications often play key roles in cell signalling and protein function, but a full account of this biochem. is far from complete. It is therefore an important goal in chem. biol. to identify what proteins are subjected to these modifications and understand their physiol. function. This review provides an overview of these modifications, how they can be detected and quantified using chem. probes, and how this information provides insight into their role in biol. This survey also highlights future opportunities in the study of cysteine redox chem., the challenges that await chemists and biologists in this area of study, and how meeting such challenges might reveal valuable information for biomedical science.
- 58Pace, N. J.; Weerapana, E. Diverse Functional Roles of Reactive Cysteines. ACS Chem. Biol. 2013, 8 (2), 283– 296, DOI: 10.1021/cb3005269[ACS Full Text], [CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1yksrfE&md5=2d27df87f153dd9c916dc901f9570644Diverse Functional Roles of Reactive CysteinesPace, Nicholas J.; Weerapana, EranthieACS Chemical Biology (2013), 8 (2), 283-296CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A review. Cysteine residues on proteins play key roles in catalysis and regulation. These functional cysteines serve as active sites for nucleophilic and redox catalysis, sites of allosteric regulation, and metal-binding ligands on proteins from diverse classes including proteases, kinases, metabolic enzymes, and transcription factors. In this review, we focus on a few select examples that serve to highlight the multiple functions performed by cysteines, with an emphasis on cysteine-mediated protein activities implicated in cancer. The enhanced reactivity of functional cysteines renders them susceptible to modification by electrophilic species. Toward this end, we discuss recent advancements and future prospects for utilizing cysteine-reactive small mols. as drugs and imaging agents for the treatment and diagnosis of cancer.
- 59Jones, A.; Zhang, X.; Lei, X. Covalent Probe Finds Carboxylic Acid. Cell Chem. Biol. 2017, 24 (5), 537– 539, DOI: 10.1016/j.chembiol.2017.05.003[Crossref], [PubMed], [CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXotlOqtLg%253D&md5=8f3451424653935cc1d168faec24c9a6Covalent Probe Finds Carboxylic AcidJones, Alexander; Zhang, Xiaoyun; Lei, XiaoguangCell Chemical Biology (2017), 24 (5), 537-539CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)In this issue of Cell Chem. Biol., Marti´n-Gago et al. (2017a) disclose a new strategy for the selective covalent targeting of binding site carboxylic acids within the proteome using the isoxazolium salt as a warhead. This discovery paves the way for developing new protein ligation methods as well as covalent drug candidates.
- 60Shirley, M. Dacomitinib: First Global Approval. Drugs 2018, 78, 1947, DOI: 10.1007/s40265-018-1028-x[Crossref], [PubMed], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3crnslGgsw%253D%253D&md5=0e035cd4b47b60f40c2af1d3c1fb5addDacomitinib: First Global ApprovalShirley MattDrugs (2018), 78 (18), 1947-1953 ISSN:.Dacomitinib (Vizimpro(®)) is an orally administered, small-molecule irreversible inhibitor of HER1 (EGFR), HER2 and HER4 that was developed by Pfizer Inc. for the treatment of solid tumours. In September 2018, dacomitinib received its first global approval, in the USA, for use in the first-line treatment of patients with metastatic NSCLC with EGFR exon 19 deletion or exon 21 L858R substitution mutations as detected by an FDA-approved test. Registration applications for the use of dacomitinib as first-line treatment for patients with EGFR-mutation-positive metastatic NSCLC have also been submitted in the EU and Japan. This article summarizes the milestones in the development of dacomitinib leading to this first approval for the first-line treatment of patients with EGFR-mutated metastatic NSCLC.
- 61Markham, A.; Dhillon, S. Acalabrutinib: First Global Approval. Drugs 2018, 78 (1), 139– 145, DOI: 10.1007/s40265-017-0852-8[Crossref], [PubMed], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFekurvF&md5=95ac3e46917e6e1b248a1a1da537f562Acalabrutinib: First Global ApprovalMarkham, Anthony; Dhillon, SohitaDrugs (2018), 78 (1), 139-145CODEN: DRUGAY; ISSN:0012-6667. (Springer International Publishing AG)Acerta Pharma is developing the Bruton's tyrosine kinase inhibitor acalabrutinib (Calquence) for the treatment of various haematol. and solid malignancies. The drug has received accelerated approval from the US FDA for the treatment of mantle cell lymphoma based on the results of a phase II study, and phase III trials in mantle cell lymphoma and chronic lymphocytic leukemia are currently underway. This article summarizes the milestones in the development of acalabrutinib leading to this first approval for mantle cell lymphoma.
- 62Forster, M.; Gehringer, M.; Laufer, S. A. Recent Advances in JAK3 Inhibition: Isoform Selectivity by Covalent Cysteine Targeting. Bioorg. Med. Chem. Lett. 2017, 27 (18), 4229– 4237, DOI: 10.1016/j.bmcl.2017.07.079[Crossref], [PubMed], [CAS], Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtl2lsb3M&md5=3c6b2f86ccd3e3f3e254bbece5fa3ac5Recent advances in JAK3 inhibition: Isoform selectivity by covalent cysteine targetingForster, Michael; Gehringer, Matthias; Laufer, Stefan A.Bioorganic & Medicinal Chemistry Letters (2017), 27 (18), 4229-4237CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Janus kinases (JAKs) are a family of four cytosolic protein kinases with a high degree of structural similarity. Due to its very restricted role in immune regulation, JAK3 was promoted as an excellent target for immunosuppression for more than a decade, but clin. validation of this concept is still elusive. During the last years, speculation arose that kinase activity of JAK1, which cooperates with JAK3 in cytokine receptor signaling, may have a dominant role over the one of JAK3. Until recently, however, this issue could not be appropriately addressed due to a lack of highly isoform-selective tool compds. With the recent resurgence of covalent drugs, targeting of a specific cysteine that distinguishes JAK3 from other JAK family members became an attractive design option. By applying this strategy, a set of JAK3 inhibitors with excellent selectivity against other JAK isoforms and the kinome was developed during the last three years and used to decipher JAK3-dependent signaling. The data obtained with these tool compds. demonstrates that selective JAK3 inhibition is sufficient to block downstream signaling. Since one of these inhibitors is currently under evaluation in phase II clin. studies against several inflammatory disorders, it will soon become apparent whether selective JAK3 inhibition translates into clin. efficacy.
- 63Garzón, B.; Oeste, C. L.; Díez-Dacal, B.; Pérez-Sala, D. Proteomic Studies on Protein Modification by Cyclopentenone Prostaglandins: Expanding Our View on Electrophile Actions. J. Proteomics 2011, 74 (11), 2243– 2263, DOI: 10.1016/j.jprot.2011.03.028[Crossref], [PubMed], [CAS], Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlKit77P&md5=b7c68bfbcdd4bcebef40a03b82703d19Proteomic studies on protein modification by cyclopentenone prostaglandins: Expanding our view on electrophile actionsGarzon, Beatriz; Oeste, Clara L.; Diez-Dacal, Beatriz; Perez-Sala, DoloresJournal of Proteomics (2011), 74 (11), 2243-2263CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)A review. Cyclopentenone prostaglandins (cyPG) are lipid mediators that participate in the mechanisms regulating inflammation and tumorigenesis. The cyPG are electrophilic compds. that act mainly through the covalent modification of cellular proteins. The stability of many cyPG-protein adducts makes them suitable for proteomic anal. Indeed, methodol. advances in recent years have allowed identifying many cyPG targets, including components of pro-inflammatory transcription factors, cytoskeletal proteins, signaling kinases and proteins involved in redox control. Insight into the diversity of cyPG targets is providing a better understanding of their mechanism of action, uncovering novel links between resoln. of inflammation, proliferation and redox regulation. Moreover, identification of the target residues has unveiled the selectivity of protein modification by these electrophiles, providing valuable information for potential pharmacol. applications. Among the challenges ahead, the detection of proteins modified by endogenous cyPG and the quant. aspects of the modification require further efforts. Importantly, only a few years after the appearance of the first proteomic studies, research on cyPG targets is yielding new paradigms for redox and electrophilic signaling.
- 64Zhao, Z.; Liu, Q.; Bliven, S.; Xie, L.; Bourne, P. E. Determining Cysteines Available for Covalent Inhibition Across the Human Kinome. J. Med. Chem. 2017, 60 (7), 2879– 2889, DOI: 10.1021/acs.jmedchem.6b01815[ACS Full Text], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXkvVGjsLg%253D&md5=22ea95ce38eb0836d32c1332dba54fadDetermining Cysteines Available for Covalent Inhibition Across the Human KinomeZhao, Zheng; Liu, Qingsong; Bliven, Spencer; Xie, Lei; Bourne, Philip E.Journal of Medicinal Chemistry (2017), 60 (7), 2879-2889CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Covalently bound protein kinase inhibitors have been frequently designed to target non-catalytic Cys residues at the ATP binding site. Thus, it is important to know if a given Cys residue can form a covalent bond. Here, we combined a function-site interaction fingerprint method and DFT calcns. to det. the potential of Cys residues to form a covalent interaction with an inhibitor. By harnessing the human structural kinome, a comprehensive structure-based binding site Cys dataset was assembled. The orientation of the Cys SH group indicated which Cys residues could potentially form covalent bonds. These covalent inhibitor accessible Cys residues were located within 5 kinase regions (P-loop, roof of pocket, front pocket, catalytic-2 of the catalytic loop and DFG-3 close to the DFG peptide). In an independent test set, these Cys residues covered 95% of covalent kinase inhibitors. Thus, this study provides new insights into Cys reactivity and preference which is important for the prospective development of covalent kinase inhibitors.
- 65Günther, M.; Juchum, M.; Kelter, G.; Fiebig, H.; Laufer, S. Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S Mutant. Angew. Chem., Int. Ed. 2016, 55 (36), 10890– 10894, DOI: 10.1002/anie.201603736[Crossref], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1GmtbzK&md5=c3909faa7f781c2a3679f75bd3b6c5acLung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S MutantGuenther, Marcel; Juchum, Michael; Kelter, Gerhard; Fiebig, Heiner; Laufer, StefanAngewandte Chemie, International Edition (2016), 55 (36), 10890-10894CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The treatment of non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) inhibitors is made challenging by acquired resistance caused by somatic mutations. Third-generation EGFR inhibitors have been designed to overcome resistance through covalent binding to the Cys 797 residue of the enzyme, and these inhibitors are effective against most clin. relevant EGFR mutants. However, the high dependence of these recent EGFR inhibitors on this particular interaction means that addnl. mutation of Cys 797 results in poor inhibitory activity, which leads to tumor relapse in initially responding patients. A new generation of irreversible and reversible mutant EGFR inhibitors was developed with strong noncovalent binding properties, and these compds. show high inhibitory activities against the cysteine-mutated L858R/T790M/C797S EGFR.
- 66Niessen, S.; Dix, M. M.; Barbas, S.; Potter, Z. E.; Lu, S.; Brodsky, O.; Planken, S.; Behenna, D.; Almaden, C.; Gajiwala, K. S.; Ryan, K.; Ferre, R.; Lazear, M. R.; Hayward, M. M.; Kath, J. C.; Cravatt, B. F. Proteome-Wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors. Cell Chem. Biol. 2017, 24, 1388– 1400, DOI: 10.1016/j.chembiol.2017.08.017[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFOrtrnL&md5=dc2db6d29932ffdb991027ba272bd149proteome-wide map of targets of t790m-egfr-directed covalent inhibitorsNiessen, Sherry; Dix, Melissa M.; Barbas, Sabrina; Potter, Zachary E.; Lu, Shuyan; Brodsky, Oleg; Planken, Simon; Behenna, Douglas; Almaden, Chau; Gajiwala, Ketan S.; Ryan, Kevin; Ferre, RoseAnn; Lazear, Michael R.; Hayward, Matthew M.; Kath, John C.; Cravatt, Benjamin F.Cell Chemical Biology (2017), 24 (11), 1388-1400.e7CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chem. proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chem. proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety.
- 67Blewett, M. M.; Xie, J.; Zaro, B. W.; Backus, K. M.; Altman, A.; Teijaro, J. R.; Cravatt, B. F. Chemical Proteomic Map of Dimethyl Fumarate–Sensitive Cysteines in Primary Human T Cells. Sci. Signaling 2016, 9 (445), rs10, DOI: 10.1126/scisignal.aaf7694
- 68Deeks, E. D. Ibrutinib: A Review in Chronic Lymphocytic Leukaemia. Drugs 2017, 77 (2), 225– 236, DOI: 10.1007/s40265-017-0695-3[Crossref], [PubMed], [CAS], Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht12lurg%253D&md5=01ea759b722431401e1e1bb85e1f87c8Ibrutinib: A Review in Chronic Lymphocytic LeukaemiaDeeks, Emma D.Drugs (2017), 77 (2), 225-236CODEN: DRUGAY; ISSN:0012-6667. (Springer International Publishing AG)Ibrutinib (Imbruvica) is an oral irreversible inhibitor of Bruton's tyrosine kinase, a B-cell receptor (BCR) signalling kinase expressed by various hematopoietic cells, B-cell lymphomas and leukemias. The drug is indicated for the treatment of certain haematol. malignancies, including chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), which are the focus of this review. In phase III CLL/SLL trials, ibrutinib monotherapy was more effective than chlorambucil in the first-line treatment of elderly patients (RESONATE-2) and more effective than ofatumumab in previously-treated adults (RESONATE). Likewise, a combination of ibrutinib, bendamustine and rituximab was more effective in previously-treated adults than bendamustine plus rituximab in a phase III placebo-controlled study (HELIOS). These ibrutinib regimens were assocd. with significantly better progression-free survival, overall response rates, and overall survival than the comparators (in protocol-specified or planned analyses), with ibrutinib therapy providing benefit regardless of adverse prognostic factors, such as del(17p)/TP53 mutation and del(11q). Ibrutinib has an acceptable tolerability profile, although certain adverse events (e.g. bleeding and atrial fibrillation) require consideration. Redistribution lymphocytosis can occur, but is not indicative of disease progression. Although longer-term data would be beneficial, ibrutinib is a welcome treatment option for patients with CLL, including those who have higher-risk disease or are less phys. fit. Indeed, current EU and US guidelines recommend/prefer the drug for the first- and/or subsequent-line treatment of certain patients, including those with del(17p)/TP53 mutation.
- 69Bender, A. T.; Gardberg, A.; Pereira, A.; Johnson, T.; Wu, Y.; Grenningloh, R.; Head, J.; Morandi, F.; Haselmayer, P.; Liu-Bujalski, L. Ability of Bruton’s Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but Not B-Cell Receptor Signaling. Mol. Pharmacol. 2017, 91 (3), 208– 219, DOI: 10.1124/mol.116.107037[Crossref], [PubMed], [CAS], Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpsFajtr8%253D&md5=f869d52d6bc1aabf352988787ff1cbcfAbility of Bruton's tyrosine kinase inhibitors to sequester Y551 and prevent phosphorylation determines potency for inhibition of Fc receptor but not B-cell receptor signalingBender, Andrew T.; Gardberg, Anna; Pereira, Albertina; Johnson, Theresa; Wu, Yin; Grenningloh, Roland; Head, Jared; Morandi, Federica; Haselmayer, Philipp; Liu-Bujalski, LesleyMolecular Pharmacology (2017), 91 (3), 208-219CODEN: MOPMA3; ISSN:1521-0111. (American Society for Pharmacology and Experimental Therapeutics)Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large no. of Btk inhibitors have been developed. These compds. have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a mol. level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compds. can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcγR signaling as Y551 sequestering compds. were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in detg. potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compds. with a broader inhibitory profile that will likely result in greater efficacy.
- 70Pan, Z.; Scheerens, H.; Li, S.-J.; Schultz, B. E.; Sprengeler, P. A.; Burrill, L. C.; Mendonca, R. V.; Sweeney, M. D.; Scott, K. C. K.; Grothaus, P. G.; Jeffery, D. A.; Spoerke, J. M.; Honigberg, L. A.; Young, P. R.; Dalrymple, S. A.; Palmer, J. T. Discovery of Selective Irreversible Inhibitors for Bruton’s Tyrosine Kinase. ChemMedChem 2007, 2 (1), 58– 61, DOI: 10.1002/cmdc.200600221[Crossref], [PubMed], [CAS], Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjsVWjsbg%253D&md5=023ba8bca9942ad0127d7cf32293e9d2Discovery of selective irreversible inhibitors for bruton's tyrosine kinasePan, Zhengying; Scheerens, Heleen; Li, Shyr-Jiann; Schultz, Brian E.; Sprengeler, Paul A.; Burrill, L. Chuck; Mendonca, Rohan V.; Sweeney, Michael D.; Scott, Keana C. K.; Grothaus, Paul G.; Jeffery, Douglas A.; Spoerke, Jill M.; Honigberg, Lee A.; Young, Peter R.; Dalrymple, Stacie A.; Palmer, James T.ChemMedChem (2007), 2 (1), 58-61CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)Synthesis and pharmacol. evaluation of a series of peptidomimetic quinoline derivs. was undertaken to evaluate their efficacy in acting as selective irreversible inhibitors of Bruton's tyrosine kinase (Btk). With the relative scarcity of knowledge on the inhibition of Btk it is crucial to discover a potent and selective tool compd. for this kinase. Herein is described the discovery of selective irreversible Btk inhibitors and their efficacy in a mouse RA model.
- 71Mann, M. Innovations: Functional and Quantitative Proteomics Using SILAC. Nat. Rev. Mol. Cell Biol. 2006, 7 (12), 952– 958, DOI: 10.1038/nrm2067[Crossref], [PubMed], [CAS], Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1KisrrF&md5=4e658b29b1c6e91abf33f86babaf93c1Functional and quantitative proteomics using SILACMann, MatthiasNature Reviews Molecular Cell Biology (2006), 7 (12), 952-958CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Researchers in many biol. areas now routinely characterize proteins by mass spectrometry. Among the many formats for quant. proteomics, stable-isotope labeling by amino acids in cell culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quant. phosphoproteomics technol. - directly uncovers important points in the signalling pathways that control cellular decisions.
- 72Crow, J. A.; Bittles, V.; Borazjani, A.; Potter, P. M.; Ross, M. K. Covalent Inhibition of Recombinant Human Carboxylesterase 1 and 2 and Monoacylglycerol Lipase by the Carbamates JZL184 and URB597. Biochem. Pharmacol. 2012, 84 (9), 1215– 1222, DOI: 10.1016/j.bcp.2012.08.017[Crossref], [PubMed], [CAS], Google Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlertLjF&md5=02eb736350283cc8b9e6224cd9f3351dCovalent inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by the carbamates JZL184 and URB597Crow, J. Allen; Bittles, Victoria; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.Biochemical Pharmacology (2012), 84 (9), 1215-1222CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metab. of several pharmaceuticals. Recently, carbamate compds. were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resp.; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate consts. for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimol. rate consts. (kinact/Ki) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 103 M-1 s-1 and 4.5 (±1.3) × 103 M-1 s-1, resp.]. However, kinact/Ki for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 102 M-1 s-1 and 3.9 (±1.0) × 103 M-1 s-1, resp.]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also detd. kinetic consts. for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metab.
- 73Buynak, J. D.; Mathew, J.; Rao, M. N.; Haley, E.; George, C.; Siriwardane, U. The Preparation of the First α-Vinylidene-β-Lactams. J. Chem. Soc., Chem. Commun. 1987, 0 (10), 735– 737, DOI: 10.1039/C39870000735[Crossref], [CAS], Google Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXlsFOjug%253D%253D&md5=d65839405f355c0236d626cd95eeb012The preparation of the first α-vinylidene-β-lactamsBuynak, John D.; Mathew, Jacob; Rao, M. Narayana; Haley, Elizabeth; George, Christian; Siriwardane, UpaliJournal of the Chemical Society, Chemical Communications (1987), (10), 735-7CODEN: JCCCAT; ISSN:0022-4936.α-Vinylidene-β-lactams I (R = H, R1 = Me, CH2Ph; R = Me, CH2Ph, CH2C6H4Br-4, CH2C6H4OMe-4, H, R1 = Me) were prepd. from MeCH:C:CHSC6H4Cl-4 and chlorosulfonyl isocyanate. Crystal structure of I (R = H, R1 = CH2Ph) was reported. β-Lactam II was prepd. similarly.
- 74Roedig, A.; Ritschel, W. Reaktionen von 3,4,4-Trichlor-3-butenamiden mit Nucleophilen, II. Thiol- und Aminaddukte von 3,3-Dichlorallencarboxamiden. Chem. Ber. 1983, 116 (4), 1595– 1602, DOI: 10.1002/cber.19831160434[Crossref], [CAS], Google Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXktVGitLc%253D&md5=7ac804c6a6bfcb66fff16d78f4968336Reactions of 3,4,4-trichloro-3-butenamides with nucleophiles. II. Thiol- and amine adducts of 3,3-dichloroallenecarboxamidesRoedig, Alfred; Ritschel, WernerChemische Berichte (1983), 116 (4), 1595-602CODEN: CHBEAM; ISSN:0009-2940.Treatment of Cl2C:C:CHCONHR (I, R = 4-MeC6H4, Me3C, Me2CH), prepd. by dehydrochlorination of Cl2C:CClCH2CONHR, with R1R2NH (R1 = R2 = Me, Et; R1 = Me, R2 = Ph; R1 = Me3C, Me2CH, Pr, R2 = H; R1R2N = morpholino) gave the corresponding adducts Cl2CHC(NR1R2):CHCONHR. Treatment of I with R3S- (R3 = 4-ClC6H4, C6Cl5, Pr, Me3C) gave Cl2C:C(SR3)CH2CONHR (II) of which II (R = Me3C, R3 = Pr, Me3C) rearrange slowly in base to give Cl2CHC(SR3):CHCONHR.
- 75Abbas, A.; Xing, B.; Loh, T.-P. Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins. Angew. Chem., Int. Ed. 2014, 53 (29), 7491– 7494, DOI: 10.1002/anie.201403121[Crossref], [CAS], Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXovVKltb0%253D&md5=bd00e8d70e768cf9a4b84422b2ee10edAllenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and ProteinsAbbas, Ata; Xing, Bengang; Loh, Teck-PengAngewandte Chemie, International Edition (2014), 53 (29), 7491-7494CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)In this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aq. medium with excellent selectivity and quant. conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biol. systems. Addnl., fluorescent-labeling studies demonstrated that the installation of a C-terminal allenamide moiety onto various mols. of interest may supply a new methodol. towards the site-specific labeling of cysteine-contg. proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.
- 76Pedzisa, L.; Li, X.; Rader, C.; Roush, W. R. Assessment of Reagents for Selenocysteine Conjugation and the Stability of Selenocysteine Adducts. Org. Biomol. Chem. 2016, 14 (22), 5141– 5147, DOI: 10.1039/C6OB00775A[Crossref], [PubMed], [CAS], Google Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XotF2msL4%253D&md5=3f96465dfc64746df2ef4704ad767dc9Assessment of reagents for selenocysteine conjugation and the stability of selenocysteine adductsPedzisa, Lee; Li, Xiuling; Rader, Christoph; Roush, William R.Organic & Biomolecular Chemistry (2016), 14 (22), 5141-5147CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Conventional antibody-drug conjugates (ADCs) are heterogeneous mixts. that have poor pharmacokinetic properties and decreased efficacy relative to homogenous ADCs. Furthermore, ADCs that are maleimide-based often have inadequate circulatory stability, which can result in premature drug release with consequent off-target toxicities. Selenocysteine-modified antibodies have been developed that allow site-specific antibody conjugation, yielding homogeneous ADCs. Herein, we survey several electrophilic functional groups that react with selenocystine with high efficiency. Several of these result in conjugates with stabilities that are superior to maleimide conjugates. Among these, the allenamide functional group reacts with notably high efficiency, leads to conjugates with remarkable stability, and shows exquisite selectivity for selenocysteine conjugation.
- 77Chen, D.; Guo, D.; Yan, Z.; Zhao, Y. Allenamide as a Bioisostere of Acrylamide in the Design and Synthesis of Targeted Covalent Inhibitors. MedChemComm 2018, 9 (2), 244– 253, DOI: 10.1039/C7MD00571G[Crossref], [PubMed], [CAS], Google Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFGns7rO&md5=919f67c6ee52d1996f0e5ccdfbe77279Allenamide as a bioisostere of acrylamide in the design and synthesis of targeted covalent inhibitorsChen, Deheng; Guo, Dexiang; Yan, Ziqin; Zhao, YujunMedChemComm (2018), 9 (2), 244-253CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)The success of acrylamide-contg. drugs in treating cancers has spurred a passion to search for acrylamide bioisosteres. In our endeavour, we have identified that an allenamide group can be a reactive bioisostere of the acrylamide group. In our development of allenamide-contg. compds., we found that the most potent compd., 14, inhibited the kinase activities of both T790M/L858R double mutant and wild type EGFR in a low nM range. 14 also inhibited the growth of NCI-H1975 lung cancer cells at IC50 = 33 nM, which is comparable to that of acrylamide-contg. osimertinib. The western blot anal. showed that the phosphorylation of EGFR, AKT, and ERK1/2 was simultaneously inhibited in a dose-dependent manner when NCI-H1975 cells were treated with 14. By measuring the conjugate addn. product formed by 14 and GSH, we obtained a reaction rate const. of 302.5 × 10-3 min-1, which is about 30-fold higher than that of osimertinib. Taken together, our data suggest that the allenamide-contg. compds. inhibited EGFR kinases through covalent modifications. Our study indicates that the allenamide group could serve as an alternative electrophilic warhead in the design of targeted covalent inhibitors, and this bioisostere replacement may have broad applications in medicinal chem.
- 78Awoonor-Williams, E.; Rowley, C. N. How Reactive Are Druggable Cysteines in Protein Kinases?. J. Chem. Inf. Model. 2018, 58 (9), 1935– 1946, DOI: 10.1021/acs.jcim.8b00454[ACS Full Text], [CAS], Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFejsLrN&md5=9ebb19e671ee3a29af129733d5803544How Reactive are Druggable Cysteines in Protein Kinases?Awoonor-Williams, Ernest; Rowley, Christopher N.Journal of Chemical Information and Modeling (2018), 58 (9), 1935-1946CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)Targeted covalent-inhibitors (TCIs) have been successfully developed as high-affinity and selective inhibitors of enzymes of the protein kinase family. These drugs typically act by undergoing an electrophilic addn. with an active site cysteine residue, so design of a TCI begins with the identification of a "druggable" cysteine. These electrophilic addns. generally require the deprotonation of the thiol to form a reactive anionic thiolate, so the acidity of the residue is a crit. factor. Few exptl. measurements of the pKa's of druggable cysteines have been reported, so computational prediction could prove to be very important in selecting reactive cysteine targets. Here, we report the computed pKa's of druggable cysteines in select protein kinases which are of clin. relevance for targeted therapies. The pKa's of the cysteines were calcd. using advanced computational methods based on all-atom replica-exchange thermodn. integration mol. dynamics simulations in explicit solvent. We found that the acidities of druggable cysteines within protein kinases are diverse and elevated, indicating enormous differences in their reactivity. Const. pH mol. dynamics simulations were also performed on select protein kinases, with results confirming this varied range in the acidities of druggable cysteines. Many of these active site cysteines have low exposure to solvent mols., elevating their pKa. Electrostatic interactions with nearby anionic residues also elevate the pKa's of cysteine residues in the active site. The results suggest that some cysteine residues within kinase binding sites will be slow to react with a TCI due to their low acidity. Several oncogenic kinase mutations were also modeled and found to have similar pKa's to the wild-type.
- 79Koniev, O.; Leriche, G.; Nothisen, M.; Remy, J.-S.; Strub, J.-M.; Schaeffer-Reiss, C.; Van Dorsselaer, A.; Baati, R.; Wagner, A. Selective Irreversible Chemical Tagging of Cysteine with 3-Arylpropiolonitriles. Bioconjugate Chem. 2014, 25 (2), 202– 206, DOI: 10.1021/bc400469d[ACS Full Text], [CAS], Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtl2nsQ%253D%253D&md5=6a55f6f7c3ca774016d6c92c5e21812cSelective Irreversible Chemical Tagging of Cysteine with 3-ArylpropiolonitrilesKoniev, Oleksandr; Leriche, Geoffray; Nothisen, Marc; Remy, Jean-Serge; Strub, Jean-Marc; Schaeffer-Reiss, Christine; Van Dorsselaer, Alain; Baati, Rachid; Wagner, AlainBioconjugate Chemistry (2014), 25 (2), 202-206CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Exquisite chemoselectivity for cysteine has been found for a novel class of remarkably hydrolytically stable reagents, 3-arylpropiolonitriles (APN). The efficacy of the APN-mediated tagging was benchmarked against other cysteine-selective methodologies in a model study on a series of traceable amino acid derivs. The selectivity of the methodol. was further explored on peptide mixts. obtained by trypsin digestion of lysozyme. Addnl., the superior stability of APN-cysteine conjugates in aq. media, human plasma, and living cells makes this new thiol-click reaction a promising methodol. for applications in bioconjugation.
- 80Shiu, H.-Y.; Chan, T.-C.; Ho, C.-M.; Liu, Y.; Wong, M.-K.; Che, C.-M. Electron-Deficient Alkynes as Cleavable Reagents for the Modification of Cysteine-Containing Peptides in Aqueous Medium. Chem. - Eur. J. 2009, 15 (15), 3839– 3850, DOI: 10.1002/chem.200800669[Crossref], [PubMed], [CAS], Google Scholar80https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksleitr0%253D&md5=cdf4acf18e4737c673a807ed1968aae6Electron-deficient alkynes as cleavable reagents for the modification of cysteine-containing peptides in aqueous mediumShiu, Hoi-Yan; Chan, Tak-Chung; Ho, Chi-Ming; Liu, Yungen; Wong, Man-Kin; Che, Chi-MingChemistry - A European Journal (2009), 15 (15), 3839-3850CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)An efficient method has been developed for the chemoselective cysteine modification of unprotected peptides and proteins in aq. media through the formation of a vinyl sulfide linkage by using electron-deficient alkynes, including alkynoic amides, esters and alky-nones. The terminal alky-none-modified peptides could be converted back into the unmodified peptides (81% isolated yield) by adding thiols under mild conditions. The usefulness of this thiol-assisted cleavage of the vinyl sulfide linkage in peptides has been exemplified by the enrichment of a cysteine-contg. peptide (71% recovery) from a mixt. of cysteine-contg. and non-cysteine-contg. peptides.
- 81Friedman, M.; Wall, J. S. Additive Linear Free-Energy Relationships in Reaction Kinetics of Amino Groups with α,β-Unsaturated Compounds. J. Org. Chem. 1966, 31 (9), 2888– 2894, DOI: 10.1021/jo01347a036[ACS Full Text], [CAS], Google Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF28Xkslert7k%253D&md5=478d318c5d9ffb47182f119daf52fac3Additive linear free-energy relationships in reaction kinetics of amino groups with α,β-unsaturated compoundsFriedman, Mendel; Wall, Joseph S.Journal of Organic Chemistry (1966), 31 (9), 2888-94CODEN: JOCEAH; ISSN:0022-3263.Reaction rates of nucleophilic addns. of amino groups in amino acids and peptides to α,β-unsatd. compds. were studied as functions of structural variations in both reactants. The obsd. wide variations in rates are correlated by a linear free-energy equation, which includes parameters assocd. with polar and steric factors of both reactants. This equation has theor. significance and should be of value for the calcn. of predicted polar and steric factors and reaction rates of a large no. of compds. Linear relationships were obsd. between reaction rates for a no. of vinyl compds. and several physicochem. parameters inherent in structural features assocd. with these compds. These relationships indicate that resonance stabilization of ground and transition rates appears to be the main factor that governs relative electrophilic reactivities of α,β-unsatd. compds. 25 refs.
- 82Cavins, J. F.; Friedman, M. An Internal Standard for Amino Acid Analyses: S-β-(4-Pyridylethyl)-l-Cysteine. Anal. Biochem. 1970, 35 (2), 489– 493, DOI: 10.1016/0003-2697(70)90211-3[Crossref], [PubMed], [CAS], Google Scholar82https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3cXktlWgu7s%253D&md5=e2b5a8763c785a40223664b09ef4dc85Reactions of amino acids, peptides, and proteins with α,β-unsaturated compounds. XIII. Internal standard for amino acid analyses: S-β-(4-pyridylethyl)-L-cysteineCavins, James F.; Friedman, MendelAnalytical Biochemistry (1970), 35 (2), 489-93CODEN: ANBCA2; ISSN:0003-2697.The new amino acid, S-β-(4-pyridylethyl)-L-cysteine (I), was prepd. by treating L-cysteine with 4-vinylpyridine in an aq. medium containing Et3N. The postulated structure for I was confirmed by IR, NMR, and mass spectroscopic analyses. I is stable to acid under conditions used for protein hydrolysis, it elutes on a basic column as a discrete peak before arginine, and its ninhydrin color is linear with concn. The new amino acid has been evaluated as an internal std. for amino acid analyses. Equally excellent results were obtained when I was added to the protein either before or after hydrolysis. Addn. of I before hydrolysis eliminates the need for N anal. of the hydrolyzate and for accurate sample application to the column.
- 83Gill, A. L.; Frederickson, M.; Cleasby, A.; Woodhead, S. J.; Carr, M. G.; Woodhead, A. J.; Walker, M. T.; Congreve, M. S.; Devine, L. A.; Tisi, D.; O’Reilly, M.; Seavers, L. C. A.; Davis, D. J.; Curry, J.; Anthony, R.; Padova, A.; Murray, C. W.; Carr, R. A. E.; Jhoti, H. Identification of Novel P38α MAP Kinase Inhibitors Using Fragment-Based Lead Generation. J. Med. Chem. 2005, 48 (2), 414– 426, DOI: 10.1021/jm049575n[ACS Full Text], [CAS], Google Scholar83https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtFCiu7nJ&md5=a128859d7f3725f2672512331b6bdc83Identification of Novel p38α MAP Kinase Inhibitors Using Fragment-Based Lead GenerationGill, Adrian L.; Frederickson, Martyn; Cleasby, Anne; Woodhead, Steven J.; Carr, Maria G.; Woodhead, Andrew J.; Walker, Margaret T.; Congreve, Miles S.; Devine, Lindsay A.; Tisi, Dominic; O'Reilly, Marc; Seavers, Lisa C. A.; Davis, Deborah J.; Curry, Jayne; Anthony, Rachel; Padova, Alessandro; Murray, Christopher W.; Carr, Robin A. E.; Jhoti, HarrenJournal of Medicinal Chemistry (2005), 48 (2), 414-426CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We describe the structure-guided optimization of the mol. fragments 2-amino-3-benzyloxypyridine 1 (IC50 1.3 mM) and 3-(2-(4-pyridyl)ethyl)indole 2 (IC50 35 μM) identified using X-ray crystallog. screening of p38α MAP kinase. Using two sep. case studies, the article focuses on the key compds. synthesized, the structure-activity relationships and the binding mode observations made during this optimization process, resulting in two potent lead series that demonstrate significant increases in activity. We describe the process of compd. elaboration either through the growing out from fragments into adjacent pockets or through the conjoining of overlapping fragments and demonstrate that we have exploited the mobile conserved activation loop, consisting in part of Asp168-Phe169-Gly170 (DFG), to generate significant improvements in potency and kinase selectivity.
- 84Raux, E.; Klenc, J.; Blake, A.; Sączewski, J.; Strekowski, L. Conjugate Addition of Nucleophiles to the Vinyl Function of 2-Chloro-4-Vinylpyrimidine Derivatives. Molecules 2010, 15 (3), 1973– 1984, DOI: 10.3390/molecules15031973[Crossref], [PubMed], [CAS], Google Scholar84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktVymu7s%253D&md5=7795a89f928d41ec009dd2202f7f2a60Conjugate addition of nucleophiles to the vinyl function of 2-chloro-4-vinylpyrimidine derivativesRaux, Elizabeth; Klenc, Jeffrey; Blake, Ava; Saczewski, Jaroslaw; Strekowski, LucjanMolecules (2010), 15 (), 1973-1984CODEN: MOLEFW; ISSN:1420-3049. (Molecular Diversity Preservation International)Conjugate addn. reaction of various nucleophiles across the vinyl group of 2-chloro-4-vinylpyrimidine, 2-chloro-4-(1-phenylvinyl)pyrimidine and 2-chloro-4-vinylquinazoline provides the corresponding 2-chloro-4-(2-substituted ethyl)pyrimidines and 2-chloro-4-(2-substituted ethyl)quinazolines. Treatment of these products, without isolation, with N-methylpiperazine results in nucleophilic displacement of chloride and yields the corresponding 2,4-disubstituted pyrimidines, e.g., I and quinazolines, e.g., II.
- 85Burns, A. R.; Kerr, J. H.; Kerr, W. J.; Passmore, J.; Paterson, L. C.; Watson, A. J. B. Tuned Methods for Conjugate Addition to a Vinyl Oxadiazole; Synthesis of Pharmaceutically Important Motifs. Org. Biomol. Chem. 2010, 8 (12), 2777– 2783, DOI: 10.1039/c001772h[Crossref], [PubMed], [CAS], Google Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmvVWktL4%253D&md5=9a7a8ec8555b6c165d9f2eaa6c1eb8b5Tuned methods for conjugate addition to a vinyl oxadiazole; synthesis of pharmaceutically important motifsBurns, Alan R.; Kerr, Jennifer H.; Kerr, William J.; Passmore, Joanna; Paterson, Laura C.; Watson, Allan J. B.Organic & Biomolecular Chemistry (2010), 8 (12), 2777-2783CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)The addn. of various nucleophiles to 3-phenyl-5-vinyl-1,2,4-oxadiazole is described. Following optimization, individual protocols tuned for the use of each specific class of reagent have been developed to allow the installation of nitrogen, sulfur, oxygen, and carbon nucleophiles, and leading to the prepn. of a series of compds. contg. the pharmaceutically important oxadiazole motif.
- 86Kuchař, M.; Hocek, M.; Pohl, R.; Votruba, I.; Shih, I.; Mabery, E.; Mackman, R. Synthesis, Cytostatic, and Antiviral Activity of Novel 6-[2-(Dialkylamino)Ethyl]-, 6-(2-Alkoxyethyl)-, 6-[2-(Alkylsulfanyl)Ethyl]-, and 6-[2-(Dialkylamino)Vinyl]Purine Nucleosides. Bioorg. Med. Chem. 2008, 16 (3), 1400– 1424, DOI: 10.1016/j.bmc.2007.10.063[Crossref], [PubMed], [CAS], Google Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhvVWgsr8%253D&md5=1b4eaa340d0f2abc73bbbe2530b7ebddSynthesis, cytostatic, and antiviral activity of novel 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, 6-[2-(alkylsulfanyl)ethyl]-, and 6-[2-(dialkylamino)vinyl]purine nucleosidesKuchar, Martin; Hocek, Michal; Pohl, Radek; Votruba, Ivan; Shih, I-hung; Mabery, Eric; Mackman, RichardBioorganic & Medicinal Chemistry (2008), 16 (3), 1400-1424CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)An efficient and facile synthesis of a large series of diverse 6-[2-(dialkylamino)vinyl]-, 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, and 6-[2-(alkylsulfanyl)ethyl]purine nucleosides (35 examples of both ribo- and 2'-deoxyribonucleosides) was developed. The key transformations involved conjugate nucleophilic addns. of amines, alcoholates, or thiolates to Tol-protected 6-alkylylpurine or 6-vinylpurine nucleosides. 6-[(2-Dialkylamino)vinyl]- and some 6-[(2-dialkylamino)ethyl]purine ribonucleosides exerted significant cytostatic effects and some anti-HCV activity with low selectivity.
- 87Il’yasov, E. A.; Galust’yan, G. G. Homolytic Addition of 1-Alkanethiols to 5-Ethynyl-2-Methylpyridine. Chem. Heterocycl. Compd. 1999, 35 (10), 1187– 1189, DOI: 10.1007/BF02323377
- 88Wipf, P.; Graham, T. H. Synthesis and Hetero-Michael Addition Reactions of 2-Alkynyl Oxazoles and Oxazolines. Org. Biomol. Chem. 2005, 3 (1), 31– 35, DOI: 10.1039/b413604g[Crossref], [PubMed], [CAS], Google Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVylt7vE&md5=861c6c74059a0ee8a8667ea89646a201Synthesis and hetero-Michael addition reactions of 2-alkynyl oxazoles and oxazolinesWipf, Peter; Graham, Thomas H.Organic & Biomolecular Chemistry (2005), 3 (1), 31-35CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Stereoselective conjugate addns. of alcs., amines, thiols, and halides to C(2)-alkynyl oxazoles and oxazolines provide a versatile entry to heterocyclic building blocks. For example, the reaction of ethanethiol with 2-(ethynyl)-4-oxazolecarboxylic acid Me ester (I) gave 2-[(1Z)-2-(ethylthio)ethenyl]-4-oxazolecarboxylic acid Me ester (II) stereoselectively.
- 89Li, Q.-F.; Yang, Y.; Maleckis, A.; Otting, G.; Su, X.-C. Thiol–Ene Reaction: A Versatile Tool in Site-Specific Labelling of Proteins with Chemically Inert Tags for Paramagnetic NMR. Chem. Commun. 2012, 48 (21), 2704– 2706, DOI: 10.1039/c2cc17900h[Crossref], [PubMed], [CAS], Google Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XitFCgurw%253D&md5=bd028717e4192f1d5f35a96892988d7cThiol-ene reaction: a versatile tool in site-specific labelling of proteins with chemically inert tags for paramagnetic NMRLi, Qing-Feng; Yang, Yin; Maleckis, Ansis; Otting, Gottfried; Su, Xun-ChengChemical Communications (Cambridge, United Kingdom) (2012), 48 (21), 2704-2706CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Site-specific tagging of proteins with paramagnetic lanthanides generates valuable long-range structure restraints for structural biol. by NMR spectroscopy. The authors show that the thiol-ene addn. reaction offers a powerful tool for tagging proteins in a chem. stable manner with very small lanthanide tags.
- 90Yang, Y.; Li, Q.-F.; Cao, C.; Huang, F.; Su, X.-C. Site-Specific Labeling of Proteins with a Chemically Stable, High-Affinity Tag for Protein Study. Chem. - Eur. J. 2013, 19 (3), 1097– 1103, DOI: 10.1002/chem.201202495[Crossref], [PubMed], [CAS], Google Scholar90https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1OgsLrM&md5=6bab43146e47348dc0ed1a1d0fa6c718Site-Specific Labeling of Proteins with a Chemically Stable, High-Affinity Tag for Protein StudyYang, Yin; Li, Qing-Feng; Cao, Chan; Huang, Feng; Su, Xun-ChengChemistry - A European Journal (2013), 19 (3), 1097-1103CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)Site-specific labeling of proteins with paramagnetic lanthanides offers unique opportunities by virtue of NMR spectroscopy in structural biol. In particular, these paramagnetic data, generated by the anisotropic paramagnetism including pseudocontact shifts (PCS), residual dipolar couplings (RDC), and paramagnetic relaxation enhancement (PRE), are highly valuable in structure detn. and mobility studies of proteins and protein-ligand complexes. Herein, we present a new way to label proteins in a site-specific manner with a high-affinity and chem. stable tag, 4-vinyl(pyridine-2,6-diyl)bismethylenenitrilo tetrakis(acetic acid) (4VPyMTA), through thiol alkylation. Its performance has been demonstrated in G47C and E64C mutants of human ubiquitin both in vitro and in a crowded environment. In comparison with the published tags, 4VPyMTA has several interesting features: (1) it has a very high binding affinity for lanthanides (higher than EDTA), (2) there is no heterogeneity in complexes with lanthanides, (3) the derivatized protein is stable and potentially applicable to the in situ anal. of proteins.
- 91Ma, F.-H.; Chen, J.-L.; Li, Q.-F.; Zuo, H.-H.; Huang, F.; Su, X.-C. Kinetic Assay of the Michael Addition-Like Thiol–Ene Reaction and Insight into Protein Bioconjugation. Chem. - Asian J. 2014, 9 (7), 1808– 1816, DOI: 10.1002/asia.201402095[Crossref], [PubMed], [CAS], Google Scholar91https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXosV2gsro%253D&md5=e4878dfc5437061ec2b42f5098341589Kinetic Assay of the Michael Addition-Like Thiol-Ene Reaction and Insight into Protein BioconjugationMa, Fei-He; Chen, Jia-Liang; Li, Qing-Feng; Zuo, Hui-Hui; Huang, Feng; Su, Xun-ChengChemistry - An Asian Journal (2014), 9 (7), 1808-1816CODEN: CAAJBI; ISSN:1861-4728. (Wiley-VCH Verlag GmbH & Co. KGaA)The chem. modification of proteins is a valuable technique in understanding the functions, interactions, and dynamics of proteins. Reactivity and selectivity are key issues in current chem. modification of proteins. The Michael addn.-like thiol-ene reaction is a useful tool that can be used to tag proteins with high selectivity for the solvent-exposed thiol groups of proteins. To obtain insight into the bioconjugation of proteins with this method, a kinetic anal. was performed. New vinyl-substituted pyridine derivs. were designed and synthesized. The reactivity of these vinyl tags with L-cysteine was evaluated by UV absorption and high-resoln. NMR spectroscopy. The results show that protonation of pyridine plays a key role in the overall reaction rates. The kinetic parameters were assessed in protein modification. The different reactivities of these vinyl tags with solvent-exposed cysteine is valuable information in the selective labeling of proteins with multiple functional groups.
- 92Wood, E. R.; Shewchuk, L. M.; Ellis, B.; Brignola, P.; Brashear, R. L.; Caferro, T. R.; Dickerson, S. H.; Dickson, H. D.; Donaldson, K. H.; Gaul, M.; Griffin, R. J.; Hassell, A. M.; Keith, B.; Mullin, R.; Petrov, K. G.; Reno, M. J.; Rusnak, D. W.; Tadepalli, S. M.; Ulrich, J. C.; Wagner, C. D.; Vanderwall, D. E.; Waterson, A. G.; Williams, J. D.; White, W. L.; Uehling, D. E. 6-Ethynylthieno[3,2-d]- and 6-Ethynylthieno[2,3-d]Pyrimidin-4-Anilines as Tunable Covalent Modifiers of ErbB Kinases. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (8), 2773– 2778, DOI: 10.1073/pnas.0708281105[Crossref], [PubMed], [CAS], Google Scholar92https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjtVSjtbw%253D&md5=fbfeeff1f03e8f7fb2c9e228231ff92e6-Ethynylthieno[3,2-d]- and 6-ethynylthieno[2,3-d]pyrimidin-4-anilines as tunable covalent modifiers of ErbB kinasesWood, Edgar R.; Shewchuk, Lisa M.; Ellis, Byron; Brignola, Perry; Brashear, Ronald L.; Caferro, Thomas R.; Dickerson, Scott H.; Dickson, Hamilton D.; Donaldson, Kelly H.; Gaule, Michael; Griffin, Robert J.; Hassell, Anne M.; Keith, Barry; Mullin, Robert; Petrov, Kimberly G.; Reno, Michael J.; Rusnak, David W.; Tadepalli, Sarva M.; Ulrich, John C.; Wagner, Craig D.; Vanderwall, Dana E.; Waterson, Alex G.; Williams, Jon D.; White, Wendy L.; Uehling, David E.Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (8), 2773-2778CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Anal. of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678-1020) with a structurally related propargylic amine (8). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure-activity relation understanding of the covalent modifying potential and biol. activity of a series of acetylenic thienopyrimidine compds. with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.
- 93Smaill, J. B.; Rewcastle, G. W.; Loo, J. A.; Greis, K. D.; Chan, O. H.; Reyner, E. L.; Lipka, E.; Showalter, H. D. H.; Vincent, P. W.; Elliott, W. L.; Denny, W. A. Tyrosine Kinase Inhibitors. 17. Irreversible Inhibitors of the Epidermal Growth Factor Receptor: 4-(Phenylamino)Quinazoline- and 4-(Phenylamino)Pyrido[3,2-d]Pyrimidine-6-Acrylamides Bearing Additional Solubilizing Functions. J. Med. Chem. 2000, 43 (7), 1380– 1397, DOI: 10.1021/jm990482t[ACS Full Text], [CAS], Google Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhs1Crtr0%253D&md5=6c569a822c398df08559abde31a1d4acTyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4-(Phenylamino)quinazoline- and 4-(Phenylamino)pyrido[3,2-d]pyrimidine-6-acrylamides bearing additional solubilizing functionsSmaill, Jeff B.; Rewcastle, Gordon W.; Loo, Joseph A.; Greis, Kenneth D.; Chan, O. Helen; Reyner, Eric L.; Lipka, Elke; Showalter, H. D. Hollis; Vincent, Patrick W.; Elliott, William L.; Denny, William A.Journal of Medicinal Chemistry (2000), 43 (7), 1380-1397CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)4-Anilinoquinazoline- and 4-anilinopyrido[3,2-d]pyrimidine-6-acrylamides substituted with solubilizing 7-alkylamine or 7-alkoxyamine side chains were prepd. by reaction of the corresponding 6-amines with acrylic acid or acrylic acid anhydrides. In the pyrido[3,2-d]pyrimidine series, the intermediate 6-amino-7-alkylamines were prepd. from 7-bromo-6-fluoropyrido[3,2-d]pyrimidine via Stille coupling with the appropriate stannane under palladium(0) catalysis. This proved a versatile method for the introduction of cationic solubilizing side chains. The compds. were evaluated for their inhibition of phosphorylation of the isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells. Quinazoline analogs with 7-alkoxyamine solubilizing groups were potent irreversible inhibitors of the isolated EGFR enzyme, with IC50[app] values from 2 to 4 nM, and potently inhibited both EGFR and erbB2 autophosphorylation in cells. 7-Alkylamino- and 7-alkoxyaminopyrido[3,2-d]pyrimidines were also irreversible inhibitors with equal or superior potency against the isolated enzyme but were less effective in the cellular autophosphorylation assays. Both quinazoline- and pyrido[3,2-d]pyrimidine-6-acrylamides bound at the ATP site alkylating cysteine 773, as shown by electrospray ionization mass spectrometry, and had similar rates of absorptive and secretory transport in Caco-2 cells. A comparison of two 7-propoxymorpholide analogs showed that the pyrido[3,2-d]pyrimidine-6-acrylamide had greater amide instability and higher acrylamide reactivity, being converted to glutathione adducts in cells more rapidly than the corresponding quinazoline. This difference may contribute to the obsd. lower cellular potency of the pyrido[3,2-d]pyrimidine-6-acrylamides. Selected compds. showed high in vivo activity against A431 xenografts on oral dosing, with the quinazolines being superior to the pyrido[3,2-d]pyrimidines. Overall, the quinazolines proved superior to previous analogs in terms of aq. soly., potency, and in vivo antitumor activity, and one example (CI 1033) has been selected for clin. evaluation.
- 94Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.; Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.; Greenberger, L. M.; Wang, Y.-F.; Wissner, A. 6-Substituted-4-(3-Bromophenylamino)Quinazolines as Putative Irreversible Inhibitors of the Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor (HER-2) Tyrosine Kinases with Enhanced Antitumor Activity. J. Med. Chem. 2001, 44 (17), 2719– 2734, DOI: 10.1021/jm0005555[ACS Full Text], [CAS], Google Scholar94https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXltFChsb8%253D&md5=989e6e77195324d92e70ca13f532e1cf6-Substituted-4-(3-bromophenylamino)quinazolines as Putative Irreversible Inhibitors of the Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor (HER-2) Tyrosine Kinases with Enhanced Antitumor ActivityTsou, Hwei-Ru; Mamuya, Nellie; Johnson, Bernard D.; Reich, Marvin F.; Gruber, Brian C.; Ye, Fei; Nilakantan, Ramaswamy; Shen, Ru; Discafani, Carolyn; DeBlanc, Ronald; Davis, Rachel; Koehn, Frank E.; Greenberger, Lee M.; Wang, Yu-Fen; Wissner, AllanJournal of Medicinal Chemistry (2001), 44 (17), 2719-2734CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of new 6-substituted-4-(3-bromophenylamino)quinazoline derivs. that may function as irreversible inhibitors of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases have been prepd. These inhibitors have, at the C-6 position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solubilizing substituents. These compds. were prepd. by acylation of 6-amino-4-(3-bromophenylamino)quinazoline with unsatd. acid chlorides or mixed anhydrides. We show that attaching a basic functional group onto the Michael acceptor results in greater reactivity, due to intramol. catalysis of the Michael addn. and/or an inductive effect of the protonated basic group. This, along with improved water soly., results in compds. with enhanced biol. properties. We present mol. modeling and exptl. evidence that these inhibitors interact covalently with the target enzymes. One compd., (I) was shown to have excellent oral activity in a human epidermoid carcinoma (A431) xenograft model in nude mice.
- 95Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.; Johnson, B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; Shi, X.; Rabindran, S. K.; Gruber, B. C.; Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang, Y.-F.; Greenberger, L. M.; Tsou, H.-R. Synthesis and Structure–Activity Relationships of 6,7-Disubstituted 4-Anilinoquinoline-3-Carbonitriles. The Design of an Orally Active, Irreversible Inhibitor of the Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor (EGFR) and the Human Epidermal Growth Factor Receptor-2 (HER-2). J. Med. Chem. 2003, 46 (1), 49– 63, DOI: 10.1021/jm020241c[ACS Full Text], [CAS], Google Scholar95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XptFOlsbk%253D&md5=db70c2f78c62824810854295d06ef6a4Synthesis and Structure-Activity Relationships of 6,7-Disubstituted 4-Anilinoquinoline-3-carbonitriles. The Design of an Orally Active, Irreversible Inhibitor of the Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor (EGFR) and the Human Epidermal Growth Factor Receptor-2 (HER-2)Wissner, Allan; Overbeek, Elsebe; Reich, Marvin F.; Floyd, M. Brawner; Johnson, Bernard D.; Mamuya, Nellie; Rosfjord, Edward C.; Discafani, Carolyn; Davis, Rachel; Shi, Xiaoqing; Rabindran, Sridhar K.; Gruber, Brian C.; Ye, Fei; Hallett, William A.; Nilakantan, Ramaswamy; Shen, Ru; Wang, Yu-Fen; Greenberger, Lee M.; Tsou, Hwei-RuJournal of Medicinal Chemistry (2003), 46 (1), 49-63CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of 6,7-disubstituted 4-anilinoquinoline-3-carbonitriles, e.g., I, that function as irreversible inhibitors of EGFR and HER-2 kinases have been prepd. These inhibitors have, at the 6-position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solubilizing substituents. These compds. were prepd. by acylation of 6-amino-4-(arylamino)quinoline-3-carbonitriles, e.g., II, with unsatd. acid chlorides or mixed anhydrides. Competitive reactivity studies were performed, showing that attaching a dialkylamino group onto the end of the Michael acceptor results in compds. with greater reactivity due to intramol. catalysis of the Michael addn. This, along with improved water-soly. results in compds. with enhanced biol. properties. Mol. modeling results consistent with the proposed mechanism of inhibition are presented. One compd., I (EKB-569), which shows excellent oral in vivo activity, was selected for further studies and is currently in phase I clin. trials for the treatment of cancer.
- 96Hubbard, R. D.; Dickerson, S. H.; Emerson, H. K.; Griffin, R. J.; Reno, M. J.; Hornberger, K. R.; Rusnak, D. W.; Wood, E. R.; Uehling, D. E.; Waterson, A. G. Dual EGFR/ErbB-2 Inhibitors from Novel Pyrrolidinyl-Acetylenic Thieno[3,2-d]Pyrimidines. Bioorg. Med. Chem. Lett. 2008, 18 (21), 5738– 5740, DOI: 10.1016/j.bmcl.2008.09.090[Crossref], [PubMed], [CAS], Google Scholar96https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht12rsrfI&md5=ce42d0aadfaadd02bc026d781560665fDual EGFR/ErbB-2 inhibitors from novel pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidinesHubbard, Robert D.; Dickerson, Scott H.; Emerson, Holly K.; Griffin, Robert J.; Reno, Michael J.; Hornberger, Keith R.; Rusnak, David W.; Wood, Edgar R.; Uehling, David E.; Waterson, Alex G.Bioorganic & Medicinal Chemistry Letters (2008), 18 (21), 5738-5740CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)A novel class of substituted pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines has been identified that are potent and selective inhibitors of both EGFR/ErbB-2 receptor tyrosine kinases. The inhibitors are found to display a range of enzyme and cellular potency and also to display a varying level of covalent modification of the kinase targets. Selected mols., including compd. (I), were found to be potent in enzymic and cellular assays while also demonstrating exposure in the mouse from an oral dose.
- 97Stevens, K. L.; Alligood, K. J.; Alberti, J. G. B.; Caferro, T. R.; Chamberlain, S. D.; Dickerson, S. H.; Dickson, H. D.; Emerson, H. K.; Griffin, R. J.; Hubbard, R. D.; Keith, B. R.; Mullin, R. J.; Petrov, K. G.; Gerding, R. M.; Reno, M. J.; Rheault, T. R.; Rusnak, D. W.; Sammond, D. M.; Smith, S. C.; Uehling, D. E.; Waterson, A. G.; Wood, E. R. Synthesis and Stereochemical Effects of Pyrrolidinyl-Acetylenic Thieno[3,2-d]Pyrimidines as EGFR and ErbB-2 Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (1), 21– 26, DOI: 10.1016/j.bmcl.2008.11.023[Crossref], [PubMed], [CAS], Google Scholar97https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlvFKl&md5=e9fbe3252cb62022196d8424e1189ff7Synthesis and stereochemical effects of pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines as EGFR and ErbB-2 inhibitorsStevens, Kirk L.; Alligood, Krystal J.; Alberti, Jennifer G. Badiang; Caferro, Thomas R.; Chamberlain, Stanley D.; Dickerson, Scott H.; Dickson, Hamilton D.; Emerson, Holly K.; Griffin, Robert J.; Hubbard, Robert D.; Keith, Barry R.; Mullin, Robert J.; Petrov, Kimberly G.; Gerding, Roseanne M.; Reno, Michael J.; Rheault, Tara R.; Rusnak, David W.; Sammond, Douglas M.; Smith, Stephon C.; Uehling, David E.; Waterson, Alex G.; Wood, Edgar R.Bioorganic & Medicinal Chemistry Letters (2009), 19 (1), 21-26CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)A novel class of pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines, e.g., I, has been identified which potently inhibit the EGFR and ErbB-2 receptor tyrosine kinases. Synthetic modifications of the pyrrolidine carbamate moiety result in a range of effects on enzyme and cellular potency. In addn., the impact of the abs. stereochem. configuration on cellular potency and oral mouse pharmacokinetics is described.
- 98Waterson, A. G.; Petrov, K. G.; Hornberger, K. R.; Hubbard, R. D.; Sammond, D. M.; Smith, S. C.; Dickson, H. D.; Caferro, T. R.; Hinkle, K. W.; Stevens, K. L.; Dickerson, S. H.; Rusnak, D. W.; Spehar, G. M.; Wood, E. R.; Griffin, R. J.; Uehling, D. E. Synthesis and Evaluation of Aniline Headgroups for Alkynyl Thienopyrimidine Dual EGFR/ErbB-2 Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (5), 1332– 1336, DOI: 10.1016/j.bmcl.2009.01.080[Crossref], [PubMed], [CAS], Google Scholar98https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXit1Wms78%253D&md5=7ac528ab1d63f1cd6a6af3985b8034afSynthesis and evaluation of aniline headgroups for alkynyl thienopyrimidine dual EGFR/ErbB-2 kinase inhibitorsWaterson, Alex G.; Petrov, Kimberly G.; Hornberger, Keith R.; Hubbard, Robert D.; Sammond, Douglas M.; Smith, Stephon C.; Dickson, Hamilton D.; Caferro, Thomas R.; Hinkle, Kevin W.; Stevens, Kirk L.; Dickerson, Scott H.; Rusnak, David W.; Spehar, Glenn M.; Wood, Edgar R.; Griffin, Robert J.; Uehling, David E.Bioorganic & Medicinal Chemistry Letters (2009), 19 (5), 1332-1336CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Aniline 'headgroups' were synthesized and incorporated into an alkynyl thienopyrimidine series of EGFR and ErbB-2 inhibitors. Potent inhibition of enzyme activity and cellular proliferation was obsd. In certain instances, protein binding was reduced and oral exposure was found to be somewhat improved relative to compds. contg. the ref. aniline.
- 99Nijmeijer, S.; Engelhardt, H.; Schultes, S.; van de Stolpe, A. C.; Lusink, V.; de Graaf, C.; Wijtmans, M.; Haaksma, E. E. J.; de Esch, I. J. P.; Stachurski, K.; Vischer, H. F.; Leurs, R. Design and Pharmacological Characterization of VUF14480, a Covalent Partial Agonist That Interacts with Cysteine 983.36 of the Human Histamine H4 Receptor. Br. J. Pharmacol. 2013, 170, 89– 100, DOI: 10.1111/bph.12113[Crossref], [PubMed], [CAS], Google Scholar99https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtlSgtbnK&md5=dbaa12c47194793907c66f94dfcbe5fdDesign and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 983.36 of the human histamine H4 receptorNijmeijer, S.; Engelhardt, H.; Schultes, S.; van de Stolpe, A. C.; Lusink, V.; de Graaf, C.; Wijtmans, M.; Haaksma, E. E. J.; de Esch, I. J. P.; Stachurski, K.; Vischer, H. F.; Leurs, R.British Journal of Pharmacology (2013), 170 (1), 89-100CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)Background and Purpose The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E1825.46 in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C983.36 in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C983.36 by functioning as a Michael acceptor. A covalent pyrimidine analog will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research. Exptl. Approach We designed and synthesized VUF14480, and pharmacol. characterized this compd. in hH4 receptor radioligand binding, G protein activation and β-arrestin2 recruitment expts. The ability of VUF14480 to act as a covalent binder was assessed both chem. and pharmacol. Key Results VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and β-arrestin2 recruitment. VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C983.36 prevented this covalent interaction. Conclusion and Implications VUF14480 is thought to bind covalently to the hH4 receptor-C983.36 residue and partially induce hH4 receptor-mediated G protein activation and β-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH4 receptor.
- 100Schapira, A.; Bate, G.; Kirkpatrick, P. Rasagiline. Nat. Rev. Drug Discovery 2005, 4 (8), 625– 626, DOI: 10.1038/nrd1803[Crossref], [PubMed], [CAS], Google Scholar100https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmvVOqt70%253D&md5=d518736530887a0552bc4a2469ddf75cFresh from the pipeline: RasagilineSchapira, Anthony; Bate, Guy; Kirkpatrick, PeterNature Reviews Drug Discovery (2005), 4 (8), 625-626CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)There is no expanded citation for this reference.
- 101Youdim, M. B. H.; Gross, A.; Finberg, J. P. M. Rasagiline [N-Propargyl-1R(+)-Aminoindan], a Selective and Potent Inhibitor of Mitochondrial Monoamine Oxidase B. Br. J. Pharmacol. 2001, 132 (2), 500– 506, DOI: 10.1038/sj.bjp.0703826[Crossref], [PubMed], [CAS], Google Scholar101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXht1Sgu7o%253D&md5=692cfb1e7f679e073f6cbdb2c8650ba9Rasagiline [N-propargyl-1R(+)-aminoindan], a selective and potent inhibitor of mitochondrial monoamine oxidase BYoudim, Moussa B. H.; Gross, Aviva; Finberg, John P. M.British Journal of Pharmacology (2001), 132 (2), 500-506CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)Rasagiline [N-propargyl-1R(+)-aminoindan], was examd. for its monoamine oxidase (MAO) A and B inhibitor activities in rats together with its S(-)-enantiomer (TVP 1022) and the racemic compd. (AGN-1135) and compared to selegiline (1-deprenyl). The tissues that were studied for MAO inhibition were the brain, liver and small intestine. While rasagiline and AGN1135 are highly potent selective irreversible inhibitors of MAO in vitro and in vivo, the S(-) enantiomer is relatively inactive in the tissues examd. The in vitro IC50 values for inhibition of rat brain MAO activity by rasagiline are 4.43±0.92 nM (type B), and 412±123 nM (type A). The ED50 values for ex vivo inhibition of MAO in the brain and liver by a single dose of rasagiline are 0.1±0.01, 0.042±0.0045 mg kg-1 resp. for MAO-B, and 6.48±0.81, 2.38±0.35 mg kg-1 resp. for MAO-A. Selective MAO-B inhibition in the liver and brain was maintained on chronic (21 days) oral dosage with ED50 values of 0.014±0.002 and 0.013±0.001 mg kg-1 resp. The degree of selectivity of rasagiline for inhibition of MAO-B as opposed to MAO-A was similar to that of selegiline. Rasagiline was three to 15 times more potent than selegiline for inhibition of MAO-B in rat brain and liver in vivo on acute and chronic administration, but had similar potency in vitro. These data together with lack of tyramine sympathomimetic potentiation by rasagiline, at selective MAO-B inhibitory dosage, indicate that this inhibitor like selegiline may be a useful agent in the treatment of Parkinson's disease in either symptomatic or L-DOPA adjunct therapy, but lack of amphetamine-like metabolites could present a therapeutic advantage for rasagiline.
- 102Wright, A. T.; Song, J. D.; Cravatt, B. F. A Suite of Activity-Based Probes for Human Cytochrome P450 Enzymes. J. Am. Chem. Soc. 2009, 131 (30), 10692– 10700, DOI: 10.1021/ja9037609[ACS Full Text], [CAS], Google Scholar102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXot1ejsbY%253D&md5=73f0ebac489c8df2ad9cc184819f2e43A Suite of Activity-Based Probes for Human Cytochrome P450 EnzymesWright, Aaron T.; Song, Joongyu D.; Cravatt, Benjamin F.Journal of the American Chemical Society (2009), 131 (30), 10692-10700CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Cytochrome P 450 enzymes regulate a variety of endogenous signaling mols. and play central roles in the metab. of xenobiotics and drugs. We recently showed that an aryl alkyne serves as an effective activity-based probe for profiling mouse liver microsomal P450s in vitro and in vivo. However, individual P450s display distinct substrate and inhibitor specificities, indicating that multiple probe structures may be required to achieve comprehensive coverage of this large and diverse enzyme family. Here, we have synthesized a suite of P 450-directed, activity-based protein profiling (ABPP) probes that contain: (1) varied chem. architectures validated as mechanism-based inhibitors of the P 450 enzyme family, and (2) terminal alkyne groups for click chem. conjugation of reporter tags. This set of probes was screened against a wide cross-section of human P450s, leading to the discovery of an optimal set of probes that provide broad coverage of this enzyme family. We used these probes to profile the effects on P 450 activity of aromatase inhibitors in current clin. use for the treatment of breast cancer. We describe the surprising discovery that one of these aromatase inhibitors, anastrozole, significantly increases probe-labeling of P 450 1A2, indicative of a heterotypic cooperativity effect on a central P 450 isoenzyme involved in metabolizing numerous drugs and xenobiotics. The results presented herein greatly expand the suite of ABPP probes for profiling P450s and illuminate new applications for these tools to understand P 450-drug interactions.
- 103Wright, A. T.; Cravatt, B. F. Chemical Proteomic Probes for Profiling Cytochrome P450 Activities and Drug Interactions In Vivo. Chem. Biol. 2007, 14 (9), 1043– 1051, DOI: 10.1016/j.chembiol.2007.08.008[Crossref], [PubMed], [CAS], Google Scholar103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVOnsr7L&md5=a35bc551c4a2d868719fa6710e86d79eChemical Proteomic Probes for Profiling Cytochrome P450 Activities and Drug Interactions In VivoWright, Aaron T.; Cravatt, Benjamin F.Chemistry & Biology (Cambridge, MA, United States) (2007), 14 (9), 1043-1051CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)The cytochrome P 450 superfamily metabolizes many endogenous signaling mols. and drugs. P 450 enzymes are regulated by posttranslational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here we describe a chem. proteomic strategy to profile P 450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a "clickable" handle provided an activity-based probe that labels multiple P 450s both in proteomic exts. and in vivo. This probe was used to record alterations in liver P 450 activities triggered by chem. agents, including inducers of P 450 expression and direct P 450 inhibitors. The chem. proteomic strategy described herein thus offers a versatile method to monitor P 450 activities and small-mol. interactions in any biol. system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class.
- 104van Geel, R.; Pruijn, G. J. M.; van Delft, F. L.; Boelens, W. C. Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide–Alkyne Cycloaddition. Bioconjugate Chem. 2012, 23 (3), 392– 398, DOI: 10.1021/bc200365k[ACS Full Text], [CAS], Google Scholar104https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtVWhsL0%253D&md5=091ab818d0b4582a4377e6a6a305a598Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide-Alkyne Cycloadditionvan Geel, Remon; Pruijn, Ger J. M.; van Delft, Floris L.; Boelens, Wilbert C.Bioconjugate Chemistry (2012), 23 (3), 392-398CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The 1,3-dipolar cycloaddn. of azides with ring-strained alkynes is one of the few bioorthogonal reactions suitable for specific biomol. labeling in complex biol. systems. Nevertheless, azide-independent labeling of proteins by strained alkynes can occur to a varying extent, thereby limiting the sensitivity of assays based on strain-promoted azide-alkyne cycloaddn. (SPAAC). A subset of three cyclooctynes, dibenzocyclooctyne (DIBO), azadibenzocyclooctyne (DIBAC), and bicyclo[6.1.0]nonyne (BCN), was used to evaluate the azide-independent labeling of proteins in vitro. For all three cyclooctynes, thiol-yne addn. with reduced peptidylcysteines is responsible for most of the azide-independent polypeptide labeling. The identity of the reaction product was confirmed by LC-MS and NMR anal. Moreover, undesired thiol-yne reactions can be prevented by alkylating peptidylcysteine thiols with iodoacetamide (IAM). Since IAM is compatible with SPAAC, a more specific azide-dependent labeling is achieved by preincubating proteins contg. reduced cysteines with IAM.
- 105Tian, H.; Sakmar, T. P.; Huber, T. A Simple Method for Enhancing the Bioorthogonality of Cyclooctyne Reagent. Chem. Commun. 2016, 52 (31), 5451– 5454, DOI: 10.1039/C6CC01321J[Crossref], [PubMed], [CAS], Google Scholar105https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XksVaju7k%253D&md5=cc470fc660442717f7286fc528a79aedA simple method for enhancing the bioorthogonality of cyclooctyne reagentTian, He; Sakmar, Thomas P.; Huber, ThomasChemical Communications (Cambridge, United Kingdom) (2016), 52 (31), 5451-5454CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)The cross-reactivity between some cyclooctynes and thiols limits the bioorthogonality of the strain-promoted azide-alkyne cycloaddn. reaction. A low concn. of β-mercaptoethanol significantly reduces the undesirable side reaction between bicyclononyne (BCN) and cysteine and while preserving free cysteines. The authors site-specifically label a genetically encoded azido group in the visual photoreceptor rhodopsin to demonstrate the utility of the strategy.
- 106Haldón, E.; Nicasio, M. C.; Pérez, P. J. Copper-Catalysed Azide–Alkyne Cycloadditions (CuAAC): An Update. Org. Biomol. Chem. 2015, 13 (37), 9528– 9550, DOI: 10.1039/C5OB01457C[Crossref], [PubMed], [CAS], Google Scholar106https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1KmtbrE&md5=829bd0ca7fcb535afde89f531f5e3f1fCopper-catalysed azide-alkyne cycloadditions (CuAAC): an updateHaldon, Estela; Nicasio, M. Carmen; Perez, Pedro J.Organic & Biomolecular Chemistry (2015), 13 (37), 9528-9550CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)A review. The reactions of org. azides and alkynes catalyzed by copper species represent the prototypical examples of click chem were discussed in detailed. The so-called CuAAC reaction (copper-catalyzed azide-alkyne cycloaddn.), discovered in 2002, has been expanded since then to become an excellent tool in org synthesis. In this contribution the recent results described in the literature since 2010 were reviewed, classified according to the nature of the catalyst precursor: copper(I) or copper(II) salts or complexes, metallic or nano-particulated copper and several solid-supported copper systems.
- 107Ekkebus, R.; van Kasteren, S. I.; Kulathu, Y.; Scholten, A.; Berlin, I.; Geurink, P. P.; de Jong, A.; Goerdayal, S.; Neefjes, J.; Heck, A. J. R.; Komander, D.; Ovaa, H. On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases. J. Am. Chem. Soc. 2013, 135 (8), 2867– 2870, DOI: 10.1021/ja309802n[ACS Full Text], [CAS], Google Scholar107https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXit1Sqtbc%253D&md5=d0f813975b6dae3de723facd0b78e47fOn Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in ProteasesEkkebus, Reggy; van Kasteren, Sander I.; Kulathu, Yogesh; Scholten, Arjen; Berlin, Ilana; Geurink, Paul P.; de Jong, Annemieke; Goerdayal, Soenita; Neefjes, Jacques; Heck, Albert J. R.; Komander, David; Ovaa, HuibJournal of the American Chemical Society (2013), 135 (8), 2867-2870CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Active-site directed probes are powerful in studies of enzymic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of deubiquitinating enzymes (DUBs) was obsd. The resulting product was shown to be a quaternary vinyl thioether, as detd. by X-ray crystallog. Proteomic anal. of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward deubiquitinating enzymes. The obsd. reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.
- 108Sommer, S.; Weikart, N. D.; Linne, U.; Mootz, H. D. Covalent Inhibition of SUMO and Ubiquitin-Specific Cysteine Proteases by an in Situ Thiol–Alkyne Addition. Bioorg. Med. Chem. 2013, 21 (9), 2511– 2517, DOI: 10.1016/j.bmc.2013.02.039[Crossref], [PubMed], [CAS], Google Scholar108https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXksFOrt7w%253D&md5=05fcb05ee930d580e1b2d37bbc5e02ccCovalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol-alkyne additionSommer, Stefanie; Weikart, Nadine D.; Linne, Uwe; Mootz, Henning D.Bioorganic & Medicinal Chemistry (2013), 21 (9), 2511-2517CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)Posttranslational modification of proteins with ubiquitin and ubiquitin-like modifiers such as SUMO can be reverted by specific proteases, also referred to as deubiquitinases and isopeptidases, most of which are cysteine-dependent. We have found that the replacement of the conserved C-terminal glycine with propargylamine converts SUMO and ubiquitin to highly efficient covalent inhibitors of their cognate cysteine proteases. Attack of the catalytic cysteine onto the terminal alkyne results in the formation of a vinyl sulfide linkage. Although this reaction is reminiscent of the inhibitory mechanism of the isosteric nitrile inhibitors it was unexpected due to the low electrophilicity of the alkyne group. We show that a precise location of the functional group in the active site of the protease is crucial for the reaction, which was not inhibited by the presence of a radical scavenger. Furthermore, a mutational study of key catalytic residues in the SUMO-protease Senp1, that is H533A and D550A of the catalytic triad and Q597A as part of the oxyanion hole, revealed that these residues are not required for the obsd. covalent adduct formation. We therefore propose that the reaction is an in situ thiol-alkyne addn. Due to the high chem. inertness of the alkyne moiety the resp. protease inhibitors should be well-suited for cellular and therapeutic applications. In keeping with this idea, selective labeling with propargylated SUMO and Ub probes was obsd. in lysates of cell lines expressing the cognate proteases after transient transfection.
- 109Swatek, K. N.; Aumayr, M.; Pruneda, J. N.; Visser, L. J.; Berryman, S.; Kueck, A. F.; Geurink, P. P.; Ovaa, H.; van Kuppeveld, F. J. M.; Tuthill, T. J.; Skern, T.; Komander, D. Irreversible Inactivation of ISG15 by a Viral Leader Protease Enables Alternative Infection Detection Strategies. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (10), 2371– 2376, DOI: 10.1073/pnas.1710617115[Crossref], [PubMed], [CAS], Google Scholar109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1WjsbbL&md5=06a6e74508456cbca5d3449d5c60974cIrreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategiesSwatek, Kirby N.; Aumayr, Martina; Pruneda, Jonathan N.; Visser, Linda J.; Berryman, Stephen; Kueck, Anja F.; Geurink, Paul P.; Ovaa, Huib; van Kuppeveld, Frank J. M.; Tuthill, Tobias J.; Skern, Tim; Komander, DavidProceedings of the National Academy of Sciences of the United States of America (2018), 115 (10), 2371-2376CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin post-translational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the mol. basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.
- 110Arkona, C.; Rademann, J. Propargyl Amides as Irreversible Inhibitors of Cysteine Proteases—A Lesson on the Biological Reactivity of Alkynes. Angew. Chem., Int. Ed. 2013, 52 (32), 8210– 8212, DOI: 10.1002/anie.201303544[Crossref], [CAS], Google Scholar110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVeqtb0%253D&md5=18ee2b226627f5bf11f4ac54ff365884Propargyl Amides as Irreversible Inhibitors of Cysteine Proteases-A Lesson on the Biological Reactivity of AlkynesArkona, Christoph; Rademann, JoergAngewandte Chemie, International Edition (2013), 52 (32), 8210-8212CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Ubiquitin like proteins are transferred to target proteins and bound by means of isopeptide bonds in an ATP driven enzymic transfer reaction with thioester intermediates. Modified target proteins are often driven towards degrdn. De-ubiquitinating enzymes (DUB) are capable of hydrolyzing the isopeptide bonds thereby reliberating ubiquitin-like proteins and target proteins. This review discusses the role of DUBs in health and disease.
- 111Sanger, F. The Free Amino Groups of Insulin. Biochem. J. 1945, 39 (5), 507– 515, DOI: 10.1042/bj0390507[Crossref], [PubMed], [CAS], Google Scholar111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaH28XislKmsA%253D%253D&md5=7f87d7cb3a83266868814046972cde6fFree amino groups of insulinSanger, F.Biochemical Journal (1945), 39 (), 507-15CODEN: BIJOAK; ISSN:0264-6021.The free amino groups of proteins and peptides readily form derivs. with 2,4-(O2N)2C6H3F (I) at room temp. Since these derivs. are relatively stable to acid hydrolysis, hydrolysis of the substituted protein enables isolation of 2,4-dinitrophenyl amino acid derivs. which are separable by partition chromatography. This, then, forms the basis of a new method which was applied to the identification and estn. of the free amino groups in insulin. For comparison and control, several derivs. of the naturally occurring amino acids had to be synthesized. To a mixt. of 0.2 g. l-phenylalanine and 0.4 g. NaHCO3 in 5 ml. H2O was added a soln. of 0.4 g. I in 10 ml. EtOH. The mixt. was reacted 2 hrs. at room temp., giving 0.27 g. of N-2,4-dinitrophenyl-l-phenylalanine, C15H13O6N3, m. 186°. α-Acetyl-l-lysine (0.48 g.) and 0.75 g. NaHCO3 in 3 ml. H2O reacted for 4 hrs. at reflux temp. with 0.5 g. 2,4-(O2N)2C6H3Cl (II) in 10 ml. EtOH gave 0.7 g. (75%) of α-acetyl-ε-2,4-dinitrophenyl-l-lysine (III), m. 110°. III refluxed for 3 hrs. with 20% HCl gives ε-2,4-dinitrophenyl-l-lysine-HCl, which seps. from dil. HCl as the monohydrate, C12H16O6N4.HCl.H2O, m. 186°. ε-Benzoyl-l-lysine (1.25 g.) treated with 1.0 g. II gives 2 g. of α-2,4-dinitrophenyl-ε-benzoyl-l-lysine (IV). IV (0.5 g.) hydrolyzed in HOAc and HCl gave 60% of α-2,4-dinitrophenyl-l-lysine, C12H16O6N4, m. 260° (decompn.). l-Tyrosine (V) in the presence of excess I gives a compd. which is presumably O,N-bis(2,4 dinitrophenyl)-l-tyrosine (VI). Reaction of V with II gives, in addn. to VI, a compd. which was probably N-2,4-dinitrophenyl-l-tyrosine. N-Acetyl-l-tyrosine (0.55 g.) treated for 4 hrs. with 2.0 g. II gave 0.1 g. of N-acetyl-O-2,4-dinitrophenyl-l-tyrosine (VII), m. 194°. VII hydrolyzed in 20% HCl gave O-2,4-dinitrophenyl-l-tyrosine, as the hydrate, C15H13O7N3.H2O, m. 202°. On treatment of histidine with excess I only the bis(2,4-dinitrophenyl) deriv. is obtained, indicating that I reacts with the imidazole ring. This is confirmed by the slow reaction of I with α-acetylhistidine and rapid reaction with histidine Me ester to give again a bis deriv. I reacts also with SH groups of S-contg. amino acids but a mixt. of products is apparently obtained. The band rates on the chromatogram for various known 2,4-dinitrophenyl amino acids were detd. for various solvent mixts. and compared with those of the derivs. obtained from hydrolyzed dinitrophenylinsulin. No indicator was necessary since all of the derivs. were colored. Isolation of the bands enabled quant. colorimetric estn. of the derivs., using the known compds. as standards. Application of this technique shows that in an insulin submol. of mol. wt. 12,000, six free amino groups are present; 2 of these are located on glycine residues, 2 on phenylalanine residues, and 2 represent the ε-amino groups of lysine. The results suggest that the insulin submol. is made up of 4 open polypeptide chains, 2 of these having terminal glycyl residues and the other 2 terminal phenylalanyl residues, the chains being bound together most probably by -S-S- linkages.
- 112Terrier, F. Rate and Equilibrium Studies in Jackson-Meisenheimer Complexes. Chem. Rev. 1982, 82 (2), 77– 152, DOI: 10.1021/cr00048a001[ACS Full Text], [CAS], Google Scholar112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XitFKrtrY%253D&md5=47e9957a89dbb9c19be1dbe630cb8315Rate and equilibrium studies in Jackson-Meisenheimer complexesTerrier, FrancoisChemical Reviews (Washington, DC, United States) (1982), 82 (2), 77-152CODEN: CHREAY; ISSN:0009-2665.A review of the title subject and its relation to SNAr reactions with 492 refs.
- 113Terrier, F. Modern Nucleophilic Aromatic Substitution, 1st ed.; Wiley-VCH: Weinheim, 2013.
- 114Kwan, E. E.; Zeng, Y.; Besser, H. A.; Jacobsen, E. N. Concerted Nucleophilic Aromatic Substitutions. Nat. Chem. 2018, 10 (9), 917– 923, DOI: 10.1038/s41557-018-0079-7[Crossref], [PubMed], [CAS], Google Scholar114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlGiurzI&md5=4b75a2efbbd6b64587af174f07ec6d70Concerted nucleophilic aromatic substitutionsKwan, Eugene E.; Zeng, Yuwen; Besser, Harrison A.; Jacobsen, Eric N.Nature Chemistry (2018), 10 (9), 917-923CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Nucleophilic arom. substitution (SNAr) is one of the most widely applied reaction classes in pharmaceutical and chem. research, providing a broadly useful platform for the modification of arom. ring scaffolds. The generally accepted mechanism for SNAr reactions involves a two-step addn.-elimination sequence via a discrete, non-arom. Meisenheimer complex. Here the authors use 12C/13C kinetic isotope effect (KIE) studies and computational analyses to provide evidence that prototypical SNAr reactions in fact proceed through concerted mechanisms. The KIE measurements were made possible by a new technique that leverages the high sensitivity of 19F as an NMR nucleus to quantitate the degree of isotopic fractionation. This sensitive technique permits the measurement of KIEs on 10 mg of natural abundance material in one overnight acquisition. As a result, it provides a practical tool for performing detailed mechanistic analyses of reactions that form or break C-F bonds.
- 115Elbrecht, A.; Chen, Y.; Adams, A.; Berger, J.; Griffin, P.; Klatt, T.; Zhang, B.; Menke, J.; Zhou, G.; Smith, R. G.; Moller, D. E. L-764406 Is a Partial Agonist of Human Peroxisome Proliferator-Activated Receptor γ. The Role of Cys13 in Ligand Binding. J. Biol. Chem. 1999, 274 (12), 7913– 7922, DOI: 10.1074/jbc.274.12.7913[Crossref], [PubMed], [CAS], Google Scholar115https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXitFSkur8%253D&md5=11437562ebe253b2fcf27afbcc7bd597L-764406 is a partial agonist of human peroxisome proliferator-activated receptor γ. The role of Cys313 in ligand bindingElbrecht, Alex; Chen, Yuli; Adams, Alan; Berger, Joel; Griffin, Patrick; Klatt, Tracey; Zhang, Bei; Menke, John; Zhou, Gaochao; Smith, Roy G.; Moller, David E.Journal of Biological Chemistry (1999), 274 (12), 7913-7922CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Insulin-sensitizing thiazolidinedione (TZD) compds. are high affinity ligands for a member of the nuclear receptor family, peroxisome proliferator-activated receptor (PPAR) γ. A scintillation proximity assay for measurement of 3H-radiolabeled TZD binding to human PPARγ under homogeneous conditions was developed. Using this approach, a novel non-TZD compd. (L-764406) was shown to be a potent (apparent binding IC50 of 70 nM) PPARγ ligand. Preincubation of PPARγ with L-764406 prevented binding of the [3H]TZD, suggesting a covalent interaction with the receptor; in addn., structurally related analogs of L-764406, which would be predicted not to interact with PPARγ in a covalent fashion, did not displace [3H]TZD binding to PPARγ. Covalent binding of L-764406 was proven by an obsd. mol. wt. shift of a tryptic PPARγ ligand binding domain (LBD) peptide by mass spectrometric anal. A specific cysteine residue (Cys313 in helix 3 of hPPARγ2) was identified as the attachment site for this compd. In protease protection expts., the liganded receptor adopted a typical agonist conformation. L-764406 exhibited partial agonist activity in cells expressing a chimeric receptor contg. the PPARγ LBD and a cognate reporter gene and also induced the expression of the adipocyte-specific gene aP2 in 3T3-L1 cells. In contrast, L-764406 did not exhibit activity in cells transfected with chimeric receptors contg. PPARα or PPARδ LBDs. The partial agonist properties of L-764406 were also evident in a co-activator assocn. assay, indicating that the increased transcription in cells was co-activator mediated. Thus, L-764406 is a novel non-TZD ligand for PPARγ and is also the first known partial agonist for this receptor. The results suggest a crit. functional role for Cys313, and helix 3, in contributing to ligand binding and subsequent agonist-induced conformational changes.
- 116Leesnitzer, L. M.; Parks, D. J.; Bledsoe, R. K.; Cobb, J. E.; Collins, J. L.; Consler, T. G.; Davis, R. G.; Hull-Ryde, E. A.; Lenhard, J. M.; Patel, L.; Plunket, K. D.; Shenk, J. L.; Stimmel, J. B.; Therapontos, C.; Willson, T. M.; Blanchard, S. G. Functional Consequences of Cysteine Modification in the Ligand Binding Sites of Peroxisome Proliferator Activated Receptors by GW9662. Biochemistry 2002, 41 (21), 6640– 6650, DOI: 10.1021/bi0159581[ACS Full Text], [CAS], Google Scholar116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtF2htLo%253D&md5=4c8b3355a3a3b43577749d7805f85affFunctional Consequences of Cysteine Modification in the Ligand Binding Sites of Peroxisome Proliferator Activated Receptors by GW9662Leesnitzer, Lisa M.; Parks, Derek J.; Bledsoe, Randy K.; Cobb, Jeff E.; Collins, Jon L.; Consler, Thomas G.; Davis, Roderick G.; Hull-Ryde, Emily A.; Lenhard, James M.; Patel, Lisa; Plunket, Kelli D.; Shenk, Jennifer L.; Stimmel, Julie B.; Therapontos, Christina; Willson, Timothy M.; Blanchard, Steven G.Biochemistry (2002), 41 (21), 6640-6650CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-γ (PPARγ), the authors identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC50 vs. PPARγ and was 10- and 600-fold less potent in binding expts. using PPARα and PPARδ, resp. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric anal. of the PPARγ ligand binding domain treated with GW9662 established Cys285 as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARγ. The functional activity of GW9662 as an antagonist of PPARγ was confirmed in an assay of adipocyte differentiation. GW9662 showed essentially no effect on transcription when tested using both full-length PPARδ and PPARα. Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects obsd. in the reporter gene assays. Control activators increased PPAR: RXR heterodimer formation and coactivator binding to both PPARγ and PPARδ. Corepressor binding was decreased. In the case of PPARα, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding. The exptl. data indicate that GW9662 modification of each of the three PPARs results in different functional consequences. The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture expts., suggests that this compd. may be a useful tool for elucidation of the role of PPARγ in biol. processes.
- 117Shearer, B. G.; Wiethe, R. W.; Ashe, A.; Billin, A. N.; Way, J. M.; Stanley, T. B.; Wagner, C. D.; Xu, R. X.; Leesnitzer, L. M.; Merrihew, R. V.; Shearer, T. W.; Jeune, M. R.; Ulrich, J. C.; Willson, T. M. Identification and Characterization of 4-Chloro-N-(2-{[5-Trifluoromethyl)-2-Pyridyl]Sulfonyl}ethyl)Benzamide (GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) Antagonist. J. Med. Chem. 2010, 53 (4), 1857– 1861, DOI: 10.1021/jm900464j[ACS Full Text], [CAS], Google Scholar117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVGisrc%253D&md5=401381858ca225791e8a90c13bb9d5e4Identification and Characterization of 4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide (GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) AntagonistShearer, Barry G.; Wiethe, Robert W.; Ashe, Adam; Billin, Andrew N.; Way, James M.; Stanley, Thomas B.; Wagner, Craig D.; Xu, Robert X.; Leesnitzer, Lisa M.; Merrihew, Raymond V.; Shearer, Todd W.; Jeune, Michael R.; Ulrich, John C.; Willson, Timothy M.Journal of Medicinal Chemistry (2010), 53 (4), 1857-1861CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)GSK3787 (I) was identified as a potent and selective ligand for PPARδ with good pharmacokinetic properties. A detailed binding study using mass spectral anal. confirmed covalent binding to Cys249 within the PPARδ binding pocket. Gene expression studies showed that pyridylsulfone 3 antagonized the transcriptional activity of PPARδ and inhibited basal CPT1a gene transcription. Compd. 3 is a PPARδ antagonist with utility as a tool to elucidate PPARδ cell biol. and pharmacol.
- 118Babaoglu, K.; Simeonov, A.; Irwin, J. J.; Nelson, M. E.; Feng, B.; Thomas, C. J.; Cancian, L.; Costi, M. P.; Maltby, D. A.; Jadhav, A.; Inglese, J.; Austin, C. P.; Shoichet, B. K. Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase. J. Med. Chem. 2008, 51 (8), 2502– 2511, DOI: 10.1021/jm701500e[ACS Full Text], [CAS], Google Scholar118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjtFSksb0%253D&md5=7736429aeafc6b17a5d0182e24df0dd4Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-LactamaseBabaoglu, Kerim; Simeonov, Anton; Irwin, John J.; Nelson, Michael E.; Feng, Brian; Thomas, Craig J.; Cancian, Laura; Costi, M. Paola; Maltby, David A.; Jadhav, Ajit; Inglese, James; Austin, Christopher P.; Shoichet, Brian K.Journal of Medicinal Chemistry (2008), 51 (8), 2502-2511CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)High-throughput screening (HTS) is widely used in drug discovery. Esp. for screens of unbiased libraries, false positives can dominate "hit lists"; their origins are much debated. Here we det. the mechanism of every active hit from a screen of 70,563 unbiased mols. against β-lactamase using quant. HTS (qHTS). Of the 1274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting β-lactams. Mass spectra, counter-screens, and crystallog. identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to mol. docking to prioritize mols. from the same library for testing at higher concns. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 μM. These results suggest that it may be the phys. behavior of org. mols., not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens.
- 119Patterson, J. T.; Asano, S.; Li, X.; Rader, C.; Barbas, C. F. Improving the Serum Stability of Site-Specific Antibody Conjugates with Sulfone Linkers. Bioconjugate Chem. 2014, 25 (8), 1402– 1407, DOI: 10.1021/bc500276m[ACS Full Text], [CAS], Google Scholar119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht12gu77I&md5=1692372289fc0c7dabf1374851b5ccd6Improving the Serum Stability of Site-Specific Antibody Conjugates with Sulfone LinkersPatterson, James T.; Asano, Shigehiro; Li, Xiuling; Rader, Christoph; Barbas, Carlos F., IIIBioconjugate Chemistry (2014), 25 (8), 1402-1407CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Current routes for synthesizing antibody-drug conjugates commonly rely on maleimide linkers to react with cysteine thiols. However, thioether exchange with metabolites and serum proteins can compromise conjugate stability and diminish in vivo efficacy. We report the application of a phenyloxadiazole sulfone linker for the prepn. of trastuzumab conjugates. This sulfone linker site-specifically labeled engineered cysteine residues in THIOMABs and improved antibody conjugate stability in human plasma at sites previously shown to be labile for maleimide conjugates. Similarly, sulfone conjugation with selenocysteine in an anti-ROR1 scFv-Fc improved human plasma stability relative to maleimide conjugation. Kinetically controlled labeling of a THIOMAB contg. two cysteine substitutions was also achieved, offering a strategy for producing antibody conjugates with expanded valency.
- 120Zhang, D.; Devarie-Baez, N. O.; Li, Q.; Lancaster, J. R.; Xian, M. Methylsulfonyl Benzothiazole (MSBT): A Selective Protein Thiol Blocking Reagent. Org. Lett. 2012, 14 (13), 3396– 3399, DOI: 10.1021/ol301370s[ACS Full Text], [CAS], Google Scholar120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xot1antL8%253D&md5=6e785b51e6288197559db5a91a982a9aMethylsulfonyl Benzothiazole (MSBT): A Selective Protein Thiol Blocking ReagentZhang, Dehui; Devarie-Baez, Nelmi O.; Li, Qian; Lancaster, Jack R.; Xian, MingOrganic Letters (2012), 14 (13), 3396-3399CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)A new thiol blocking reagent, methylsulfonyl benzothiazole, was discovered. This reagent showed good selectivity and high reactivity for protein thiols.
- 121Toda, N.; Asano, S.; Barbas, C. F. Rapid, Stable, Chemoselective Labeling of Thiols with Julia–Kocieński-like Reagents: A Serum-Stable Alternative to Maleimide-Based Protein Conjugation. Angew. Chem., Int. Ed. 2013, 52 (48), 12592– 12596, DOI: 10.1002/anie.201306241[Crossref], [CAS], Google Scholar121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1Wms7%252FF&md5=ef91e1e933d116482b9e886c94c6cdd0Rapid, stable, chemoselective labeling of thiols with Julia-Kocienski-like reagents: A serum-stable alternative to maleimide-based protein conjugationToda, Narihiro; Asano, Shigehiro; Barbas, Carlos F., IIIAngewandte Chemie, International Edition (2013), 52 (48), 12592-12596CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)We have developed a class of sulfone derivs. for applications in protein conjugation chem., and we have compared the newly synthesized conjugates to maleimide conjugates. Methylsulfonyl-functionalized five-membered cyclic compds., such as phenyltetrazole or phenyloxadiazole, reacted rapidly and specifically with thiols in small mols. and proteins with exquisite chemoselectivity at biol. relevant pH values (pH 5.8 - 8.0). Designer heteroarom. sulfones allowed for the selective introduction of a fluorophore and poly(ethylene) glycol chains (PEGylation), and provided protein conjugates with superior stability compared to maleimide-conjugated proteins in human plasma. Given the speed, selectivity, and stability of the sulfone-cysteine reactions described herein, we anticipate that this "thiol-Click" approach will find broad application in peptide and protein chem. and for the development of antibody drug conjugates.
- 122Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y.-S.; Pentelute, B. L. A Perfluoroaryl-Cysteine SNAr Chemistry Approach to Unprotected Peptide Stapling. J. Am. Chem. Soc. 2013, 135 (16), 5946– 5949, DOI: 10.1021/ja400119t[ACS Full Text], [CAS], Google Scholar122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlsV2gtrc%253D&md5=126ac191059b0703757ef7838d90e183A Perfluoroaryl-Cysteine SNAr Chemistry Approach to Unprotected Peptide StaplingSpokoyny, Alexander M.; Zou, Yekui; Ling, Jingjing J.; Yu, Hongtao; Lin, Yu-Shan; Pentelute, Bradley L.Journal of the American Chemical Society (2013), 135 (16), 5946-5949CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors report the discovery of a facile transformation between perfluoroarom. mols. and a cysteine thiolate, which is arylated at room temp. This new approach enabled the authors to selectively modify cysteine residues in unprotected peptides, providing access to variants contg. rigid perfluoroarom. staples. This stapling modification performed on a peptide sequence designed to bind the C-terminal domain of an HIV-1 (C-CA) showed enhancement in binding, cell permeability, and proteolytic stability properties, as compared to the unstapled analog. Importantly, chem. stability of the formed staples allowed the authors to use this motif in the native chem. ligation-mediated synthesis of a small protein affibody that is capable of binding the human epidermal growth factor 2 receptor.
- 123Alapour, S.; de la Torre, B. G.; Ramjugernath, D.; Koorbanally, N. A.; Albericio, F. Application of Decafluorobiphenyl (DFBP) Moiety as a Linker in Bioconjugation. Bioconjugate Chem. 2018, 29 (2), 225– 233, DOI: 10.1021/acs.bioconjchem.7b00800[ACS Full Text], [CAS], Google Scholar123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFKjur0%253D&md5=80ad66589d3d56dd49d8b16a1a8de884Application of Decafluorobiphenyl (DFBP) Moiety as a Linker in BioconjugationAlapour, Saba; de la Torre, Beatriz G.; Ramjugernath, Deresh; Koorbanally, Neil A.; Albericio, FernandoBioconjugate Chemistry (2018), 29 (2), 225-233CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)A review. Considerable attention has been devoted to fluorinated compds. due to their unique and interesting properties. Many modern pharmaceuticals contain fluorinated substituents, which are commonly synthesized using selective fluorinating reagents. Decafluorobiphenyl (DFBP) as a fluorinated linker is susceptible to nucleophilic attack. This nucleophilic reaction has been widely studied using various nucleophiles. Sulfur and nitrogen contg. nucleophiles have been of particular interest, esp. in bioconjugated reactions. This review focuses on the SNAr reactivity of DFBP in formation of C-X (X = S, N) bonds, to be applied in bioconjugation in org. chem. The review aims to highlight the crucial factors that govern the chem. behind the activation of F-CAr-CAr-F bonds as a linker in the synthesis of novel peptides, proteins, and biologics.
- 124Brown, S. P.; Smith, A. B. Peptide/Protein Stapling and Unstapling: Introduction of s-Tetrazine, Photochemical Release, and Regeneration of the Peptide/Protein. J. Am. Chem. Soc. 2015, 137 (12), 4034– 4037, DOI: 10.1021/ja512880g[ACS Full Text], [CAS], Google Scholar124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvFWjt7w%253D&md5=55fd8c77ca0926af46e6f9a341a45008Peptide/Protein Stapling and Unstapling: Introduction of s-Tetrazine, Photochemical Release, and Regeneration of the Peptide/ProteinBrown, Stephen P.; Smith, Amos B., IIIJournal of the American Chemical Society (2015), 137 (12), 4034-4037CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protocols have been achieved that permit facile introduction of s-tetrazine into unprotected peptides and the protein, thioredoxin, between two cysteine sulfhydryl groups (i.e., staple), followed by photochem. release (i.e., unstaple) and regeneration of the peptide/ protein upon removal of the cyano groups from the derived bisthiocyanate. The S,S-tetrazine macrocycles in turn provide a convenient handle for probe introduction by exploiting the inverse electron demand Diels-Alder reactivity of the tetrazine.
- 125Roberts, D. W.; Aptula, A. O. Electrophilic Reactivity and Skin Sensitization Potency of SNAr Electrophiles. Chem. Res. Toxicol. 2014, 27 (2), 240– 246, DOI: 10.1021/tx400355n[ACS Full Text], [CAS], Google Scholar125https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjtVWmsA%253D%253D&md5=30a013cec875f8fa3d482421dada4e88Electrophilic Reactivity and Skin Sensitization Potency of SNAr ElectrophilesRoberts, D. W.; Aptula, A. O.Chemical Research in Toxicology (2014), 27 (2), 240-246CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)The authors published in 2011 a quant. mechanistic model (QMM) for skin sensitization potency of SNAr electrophiles in the mouse local lymph node assay (LLNA). In this model, potency was correlated with a combination of σ* for the leaving group and the total σ- values of the other substituents in the arom. ring. Shortly afterward Natsch et al. published a kinetic study in which rate consts. were detd. for reactions of SNAr electrophiles with the cysteine-based peptide Ac-RFAACAA (Cys-peptide) that is used in the direct peptide reactivity assay (DPRA), and correlations were sought between these rate consts. and sensitization potency in the LLNA. These two publications together have enabled the present study, aiming to develop a linear free energy relationship (LFER) correlating Cys-peptide reactivity with a reactivity parameter (RP) based on a combination of σ* and σ- substituent consts. and, by analyzing differences between the QMM based on RP and the QMM based on Cys-peptide rate consts., to gain further insights into the underlying chem. of skin sensitization. For the 2,4-dinitro-X-substituted benzenes (DNXB), the rate consts. of Natsch et al. are well correlated with the reactivity parameter used in the authors' earlier work, with two outliers. These are the compds. with X = F and X = SCN, which are both substantially more reactive toward Cys-peptide than predicted from comparison of their RP values with those of the other DNXB compds. These two compds. are both neg. outliers from a correlation of sensitization potency with exptl. rate consts., but fit well to the correlation of sensitization potency with RP values. With these two compds. excluded, sensitization potency is well correlated with the exptl. rate consts. for the DNXB compds. (X = SO3-, I, Br, Cl) together with 2,4-dichloro-1-nitrobenzene and 1,3,4,5-tetrachloro-2,6-dicyanobenzene. The regression equation is pEC3 = 0.88 log k + 4.03, R2 = 0.966. The implication of DNFB being an outlier is that the model Cys-peptide nucleophile is substantially more sterically hindered than the cutaneous nucleophile(s) involved in the sensitization process. The pattern seen with 2,4-dinitrothiocyanatobenzene suggests that this compd. reacts as an SNAr electrophile in the sensitization process, but by a different pathway, acting as a CN transfer agent, with the model Cys-peptide. For two further compds., 2,4,6-trinitrochlorobenzene and 2,4,6-trinitrobenzenesulfonate, the Cys-peptide rate consts. are well predicted by the reactivity parameter based on displacement of the Cl or SO3- substituent, but their sensitization potency is underestimated by both the Cys-peptide rate const. and this reactivity parameter. However, potency of these two compds. is well predicted by a reactivity parameter calcd. on the basis of displacement of the 2-nitro group. This is interpreted as a case of sensitization being driven by the thermodynamically favored rather than the kinetically favored reaction product.
- 126Hwang, J. Y.; Huang, W.; Arnold, L. A.; Huang, R.; Attia, R. R.; Connelly, M.; Wichterman, J.; Zhu, F.; Augustinaite, I.; Austin, C. P.; Inglese, J.; Johnson, R. L.; Guy, R. K. Methylsulfonylnitrobenzoates, a New Class of Irreversible Inhibitors of the Interaction of the Thyroid Hormone Receptor and Its Obligate Coactivators That Functionally Antagonizes Thyroid Hormone. J. Biol. Chem. 2011, 286 (14), 11895– 11908, DOI: 10.1074/jbc.M110.200436[Crossref], [PubMed], [CAS], Google Scholar126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktVKqtro%253D&md5=a968a083f7c5718c2e03d5ef0a3b25aeMethylsulfonylnitrobenzoates, a New Class of Irreversible Inhibitors of the Interaction of the Thyroid Hormone Receptor and its Obligate Coactivators that Functionally Antagonizes Thyroid HormoneHwang, Jong-Yeon; Huang, Wen-Wei; Arnold, Leggy A.; Huang, Rui-Li; Attia, Ramy R.; Connelly, Michele; Wichterman, Jennifer; Zhu, Fang-Yi; Augustinaite, Indre; Austin, Christopher P.; Inglese, James; Johnson, Ronald L.; Guy, R. KiplinJournal of Biological Chemistry (2011), 286 (14), 11895-11908CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metab. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a no. of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the assocn. of TRβ with steroid receptor coactivator 2 (SRC2), the authors identified a novel methylsulfonylnitrobenzoate (MSNB)-contg. series that blocks this interaction at micromolar concns. Here the authors have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRβ antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum anal. suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRβ to disrupt SRC2 assocn.
- 127Arnold, L. A.; Kosinski, A.; Estébanez-Perpiñá, E.; Guy, R. K. Inhibitors of the Interaction of a Thyroid Hormone Receptor and Coactivators: Preliminary Structure–Activity Relationships. J. Med. Chem. 2007, 50 (22), 5269– 5280, DOI: 10.1021/jm070556y[ACS Full Text], [CAS], Google Scholar127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFWktr%252FE&md5=396a0fbc7879935595d695c4e6c56bbaInhibitors of the Interaction of a Thyroid Hormone Receptor and Coactivators: Preliminary Structure-Activity RelationshipsArnold, Leggy A.; Kosinski, Aaron; Estebanez-Perpina, Eva; Fletterick, Robert J.; Guy, R. KiplinJournal of Medicinal Chemistry (2007), 50 (22), 5269-5280CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The modulation of gene regulation by blocking the interaction between the thyroid receptor (TR) and obligate coregulators has been reported recently with the discovery of the lead compd. 3-(dimethylamino)-1-(4-hexylphenyl)propan-1-one. Herein the authors report studies aimed at optimization of this initial hit to det. the basic parameters of the structure-activity relationships and clarify the mechanism of action. These studies provided new insights, showing that activity and TRβ isoform selectivity is highly correlated with the structural compn. of these covalent inhibitors.
- 128Visperas, P. R.; Winger, J. A.; Horton, T. M.; Shah, N. H.; Aum, D. J.; Tao, A.; Barros, T.; Yan, Q.; Wilson, C. G.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Modification by Covalent Reaction or Oxidation of Cysteine Residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding. Biochem. J. 2015, 465 (1), 149– 161, DOI: 10.1042/BJ20140793[Crossref], [PubMed], [CAS], Google Scholar128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFWiu7vE&md5=219ac232370be3247dee4e4bb7582925Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide bindingVisperas, Patrick R.; Winger, Jonathan A.; Horton, Timothy M.; Shah, Neel H.; Aum, Diane J.; Tao, Alyssa; Barros, Tiago; Yan, Qingrong; Wilson, Christopher G.; Arkin, Michelle R.; Weiss, Arthur; Kuriyan, JohnBiochemical Journal (2015), 465 (1), 149-161CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Zeta-chain assocd. protein of 70 kDa (ZAP-70) and spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signalling resp. They are recruited, via their tandem-SH2 (Src-homol. domain 2) domains, to doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their crit. roles in immune signalling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small mols. can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys39 in ZAP-70, Cys206 in Syk) that is necessary for inhibition by two of these compds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of H2O2 and these two cysteine residues are also necessary for inhibition by H2O2. Our findings suggest a mechanism by which the reactive oxygen species generated during responses to antigen could attenuate signalling through these kinases and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains.
- 129Visperas, P. R.; Wilson, C. G.; Winger, J. A.; Yan, Q.; Lin, K.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput Screen. SLAS Discov. 2017, 22 (3), 324– 331, DOI: 10.1177/1087057116681407[Crossref], [PubMed], [CAS], Google Scholar129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFKksLc%253D&md5=49eacea4b3bbba376aa7fd7785ef0e22Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput ScreenVisperas, Patrick R.; Wilson, Christopher G.; Winger, Jonathan A.; Yan, Qingrong; Lin, Kevin; Arkin, Michelle R.; Weiss, Arthur; Kuriyan, JohnSLAS Discovery (2017), 22 (3), 324-331CODEN: SDLIBT; ISSN:2472-5560. (Sage Publications)ZAP-70 is a crit. mol. in the transduction of T cell antigen receptor signaling and the activation of T cells. Upon activation of the T cell antigen receptor, ZAP-70 is recruited to the intracellular ζ-chains of the T cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T cell antigen receptor. The screen is based on a fluorescence polarization assay for peptide binding to ZAP-70.
- 130Shannon, D. A.; Banerjee, R.; Webster, E. R.; Bak, D. W.; Wang, C.; Weerapana, E. Investigating the Proteome Reactivity and Selectivity of Aryl Halides. J. Am. Chem. Soc. 2014, 136 (9), 3330– 3333, DOI: 10.1021/ja4116204[ACS Full Text], [CAS], Google Scholar130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXislentLo%253D&md5=b8859e8d24d051fb62ab4dfba7431fe1Investigating the Proteome Reactivity and Selectivity of Aryl HalidesShannon, D. Alexander; Banerjee, Ranjan; Webster, Elizabeth R.; Bak, Daniel W.; Wang, Chu; Weerapana, EranthieJournal of the American Chemical Society (2014), 136 (9), 3330-3333CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein-reactive electrophiles are crit. to chem. proteomic applications including activity-based protein profiling, site-selective protein modification, and covalent inhibitor development. Here, we explore the protein reactivity of a panel of aryl halides that function through a nucleophilic arom. substitution (SNAr) mechanism. We show that the reactivity of these electrophiles can be finely tuned by varying the substituents on the aryl ring. We identify p-chloro- and fluoronitrobenzenes and dichlorotriazines as covalent protein modifiers at low micromolar concns. Interestingly, investigating the site of labeling of these electrophiles within complex proteomes identified p-chloronitrobenzene as highly cysteine selective, whereas the dichlorotriazine favored reactivity with lysines. These studies illustrate the diverse reactivity and amino-acid selectivity of aryl halides and enable the future application of this class of electrophiles in chem. proteomics.
- 131Schardon, C. L.; Tuley, A.; Er, J. A. V.; Swartzel, J. C.; Fast, W. Selective Covalent Protein Modification by 4-Halopyridines through Catalysis. ChemBioChem 2017, 18 (15), 1551– 1556, DOI: 10.1002/cbic.201700104[Crossref], [PubMed], [CAS], Google Scholar131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKhsbrE&md5=c71e0d7b061645a374919194678cab1aSelective Covalent Protein Modification by 4-Halopyridines through CatalysisSchardon, Christopher L.; Tuley, Alfred; Er, Joyce A. V.; Swartzel, Jake C.; Fast, WalterChemBioChem (2017), 18 (15), 1551-1556CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)We have investigated 4-halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chem. probes. Nonenzymic reactivity of 4-chloropyridine with amino acids and thiols was ranked with respect to common covalent protein-modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the pos. charged pyridinium. Diverse, fragment-sized 4-halopyridines inactivated human dimethylarginine dimethylaminohydrolase-1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off-pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2-fluoromethyl-substituted 4-chloropyridines demonstrated that the pKa and kinact/KI values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4-Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.
- 132Fairhurst, R. A.; Knoepfel, T.; Leblanc, C.; Buschmann, N.; Gaul, C.; Blank, J.; Galuba, I.; Trappe, J.; Zou, C.; Voshol, J.; Genick, C.; Brunet-Lefeuvre, P.; Bitsch, F.; Graus-Porta, D.; Furet, P. Approaches to Selective Fibroblast Growth Factor Receptor 4 Inhibition through Targeting the ATP-Pocket Middle-Hinge Region. MedChemComm 2017, 8, 1604– 1613, DOI: 10.1039/C7MD00213K[Crossref], [PubMed], [CAS], Google Scholar132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXps1GlsL8%253D&md5=04f1aff88385b915e2672509dd3f6954Approaches to selective fibroblast growth factor receptor 4 inhibition through targeting the ATP-pocket middle-hinge regionFairhurst, Robin A.; Knoepfel, Thomas; Leblanc, Catherine; Buschmann, Nicole; Gaul, Christoph; Blank, Jutta; Galuba, Inga; Trappe, Jorg; Zou, Chao; Voshol, Johannes; Genick, Christine; Brunet-Lefeuvre, Peggy; Bitsch, Francis; Graus-Porta, Diana; Furet, PascalMedChemComm (2017), 8 (8), 1604-1613CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)A diverse range of selective FGFR4 inhibitor hit series were identified using unbiased screening approaches and by the modification of known kinase inhibitor scaffolds. In each case the origin of the selectivity was consistent with an interaction with a poorly conserved cysteine residue within the middle-hinge region of the kinase domain of FGFR4, at position 552. Targeting this region identified a non-covalent diaminopyrimidine series differentiating by size, an irreversible-covalent inhibitor in which Cys552 undergoes an SNAr reaction with a 2-chloropyridine, and a reversible-covalent inhibitor series in which Cys552 forms a hemithioacetal adduct with a 2-formyl naphthalene. In addn., the introduction of an acrylamide into a known FGFR scaffold identified a pan-FGFR inhibitor which reacted with both Cys552 and a second poorly conserved cysteine on the P-loop of FGFR4 at position 477 which is present in all four FGFR family members.
- 133Hou, W.; Ren, Y.; Zhang, Z.; Sun, H.; Ma, Y.; Yan, B. Novel Quinazoline Derivatives Bearing Various 6-Benzamide Moieties as Highly Selective and Potent EGFR Inhibitors. Bioorg. Med. Chem. 2018, 26 (8), 1740– 1750, DOI: 10.1016/j.bmc.2018.02.022[Crossref], [PubMed], [CAS], Google Scholar133https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjvFyntLw%253D&md5=a203ff10067b1b141637f110f0781695Novel quinazoline derivatives bearing various 6-benzamide moieties as highly selective and potent EGFR inhibitorsHou, Weijie; Ren, Yan; Zhang, Zhenhua; Sun, Huan; Ma, Yongfen; Yan, BoBioorganic & Medicinal Chemistry (2018), 26 (8), 1740-1750CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)A series of novel quinazoline derivs. bearing various C-6 benzamide substituents were synthesized and evaluated as EGFR inhibitors, and most showed significant inhibitory potency against EGFR kinase. In particular, compd. 6g possessed potent inhibitory activity against EGFR wild-type (IC50 = 5 nM), and strong antiproliferative activity against HCC827 and Ba/F3 (L858R) cell lines. Kinase profiling against a panel of 365 kinases showed that 6g was highly selective for EGFR. Furthermore, 6g showed desirable properties in assays of liver microsome metabolic stability and cytochromes P 450 inhibition and preliminary pharmacokinetic study. The overall attractive profile of 6g made it an interesting compd. for further development.
- 134Juchum, M.; Günther, M.; Laufer, S. A. Fighting Cancer Drug Resistance: Opportunities and Challenges for Mutation-Specific EGFR Inhibitors. Drug Resist. Updates 2015, 20, 12– 28, DOI: 10.1016/j.drup.2015.05.002[Crossref], [PubMed], [CAS], Google Scholar134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2Mfot1agsA%253D%253D&md5=e0f18db0613cbf9ddee3021ba7e1bc80Fighting cancer drug resistance: Opportunities and challenges for mutation-specific EGFR inhibitorsJuchum Michael; Gunther Marcel; Laufer Stefan ADrug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy (2015), 20 (), 12-28 ISSN:.Multiple mutations in the EGFR gene are a major cause for the failure of Erlotinib and Gefitinib in the treatment of patients harboring non-small-cell lung cancer (NSCLC) who initially responded to this therapy. The development of these tyrosine kinase inhibitors (TKIs) is going back to the early 90s, where cancer was widely considered and fully treated as a disease of an organ. Fundamental gain of knowledge in cell biology in general and cancer genetics in particular led us to where we currently stand: cancer is a disease that originates in the genome. Fast and affordable gene sequencing paved the way and opened our eyes for the genetic instability of many cancers, particularly EGFR driven NSCLC. This might allow highly rational and personal therapies by aiming at a very particular wild type and mutant kinase pattern. However, the paradigm "one disease - one target - one drug" is currently challenged. Both activating and deactivating EGFR mutations are known to render the development of novel targeted drugs difficult. Among all lung adenocarcinomas, only 20% are driven by EGFR and only a subpopulation has an activating mutation (e.g. L858R), making them sensitive to first generation EGFR inhibitors. Unfortunately, most of them acquire second deactivating mutations (e.g. T790M) during treatment, leading to a complete loss of response. Are specific inhibitors of the double EGFR mutant L858R/T790M the magic bullet? Much scientific evidence but also high expectations justify this approach. Structural biology of EGFR mutants constitutes the basis for highly rational approaches. Second generation pan EGFR inhibitors inhibiting wild type (WT) and mutant EGFR like Afatinib suffer from dose-limiting adverse effects. Inhibition of WT EGFR is considered to be the culprit. Third generation EGFR inhibitors follow two strategies. Mutant selectivity and improved target residential time. These inhibitors display high mutant selectivity and irreversible binding patterns while sparing WT EGFR activity, hence enhancing tumor selectivity while minimizing adverse effects. Third generation EGFR inhibitors are still undergoing preclinical and clinical evaluation. The most advanced are Rociletinib and AZD9291 which displayed encouraging preliminary clinical phase II data regarding response and adverse effects. In the current review we show both a medicinal chemists' approach toward the design of third generation EGFR inhibitors as well as a detailed overview of the development of EGFR inhibitors over the last decade. High interdisciplinary approaches, such as structural biology and time-resolved tumor genetics pave the way toward the development of drugs that target EGFR mutants. This might lead to highly effective targeted and personalized therapies with enhanced response rates for a minor cohort of patients which have to undergo continuous gene sequencing, hence enabling therapies with tailor-made TKIs.
- 135Chen, K. X.; Lesburg, C. A.; Vibulbhan, B.; Yang, W.; Chan, T.-Y.; Venkatraman, S.; Velazquez, F.; Zeng, Q.; Bennett, F.; Anilkumar, G. N.; Duca, J.; Jiang, Y.; Pinto, P.; Wang, L.; Huang, Y.; Selyutin, O.; Gavalas, S.; Pu, H.; Agrawal, S.; Feld, B.; Huang, H.-C.; Li, C.; Cheng, K.-C.; Shih, N.-Y.; Kozlowski, J. A.; Rosenblum, S. B.; Njoroge, F. G. A Novel Class of Highly Potent Irreversible Hepatitis C Virus NS5B Polymerase Inhibitors. J. Med. Chem. 2012, 55 (5), 2089– 2101, DOI: 10.1021/jm201322r[ACS Full Text], [CAS], Google Scholar135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntFGlsg%253D%253D&md5=2da02f6d3c59719f0a1b7646f1ef60cfA Novel Class of Highly Potent Irreversible Hepatitis C Virus NS5B Polymerase InhibitorsChen, Kevin X.; Lesburg, Charles A.; Vibulbhan, Bancha; Yang, Weiying; Chan, Tin-Yau; Venkatraman, Srikanth; Velazquez, Francisco; Zeng, Qingbei; Bennett, Frank; Anilkumar, Gopinadhan N.; Duca, Jose; Jiang, Yueheng; Pinto, Patrick; Wang, Li; Huang, Yuhua; Selyutin, Oleg; Gavalas, Stephen; Pu, Haiyan; Agrawal, Sony; Feld, Boris; Huang, Hsueh-Cheng; Li, Cheng; Cheng, Kuo-Chi; Shih, Neng-Yang; Kozlowski, Joseph A.; Rosenblum, Stuart B.; Njoroge, F. GeorgeJournal of Medicinal Chemistry (2012), 55 (5), 2089-2101CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Starting from indole-based C-3 pyridone HCV NS5B polymerase inhibitor I, structure-activity relationship (SAR) investigations of the indole N-1 benzyl moiety were performed. This study led to the discovery of irreversible inhibitors with p-fluoro-sulfone- or p-fluoro-nitro-substituted N-1 benzyl groups which achieved breakthrough replicon assay potency (EC50 = 1 nM). The formation of a covalent bond with adjacent cysteine-366 thiol was was proved by mass spectroscopy and X-ray crystal structure studies. The C-5 Et C-2 carboxylic acid deriv. II had an excellent oral area-under-the-curve (AUC) of 18 μM·h (10 mg/kg). Its oral exposure in monkeys and dogs was also very good. The NMR ALARM assay, mass spectroscopy expts., in vitro counter screening, and toxicol. assays demonstrated that the covalent bond formation between compd. II and the protein was highly selective and specific. The overall excellent profile of II made it an interesting candidate for further investigation.
- 136Powers, J. P.; Piper, D. E.; Li, Y.; Mayorga, V.; Anzola, J.; Chen, J. M.; Jaen, J. C.; Lee, G.; Liu, J.; Peterson, M. G.; Tonn, G. R.; Ye, Q.; Walker, N. P. C.; Wang, Z. SAR and Mode of Action of Novel Non-Nucleoside Inhibitors of Hepatitis C NS5b RNA Polymerase. J. Med. Chem. 2006, 49 (3), 1034– 1046, DOI: 10.1021/jm050859x[ACS Full Text], [CAS], Google Scholar136https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XksV2ltA%253D%253D&md5=722ac0edb5dabb720e30475ef5925d5fSAR and Mode of Action of Novel Non-Nucleoside Inhibitors of Hepatitis C NS5b RNA PolymerasePowers, Jay P.; Piper, Derek E.; Li, Yang; Mayorga, Veronica; Anzola, John; Chen, James M.; Jaen, Juan C.; Lee, Gary; Liu, Jinqian; Peterson, M. Greg; Tonn, George R.; Ye, Qiuping; Walker, Nigel P. C.; Wang, ZhulunJournal of Medicinal Chemistry (2006), 49 (3), 1034-1046CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochem. potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases. The structures of the complexes formed between several of these inhibitors and HCV NS5b were detd. by x-ray crystallog., and the inhibitors were found to bind in an allosteric binding site sep. from the active site. Structure-activity relationships and structural studies have identified the mechanism of action for compds. in this series, several of which possess drug-like properties, as unique, reversible, covalent inhibitors of HCV NS5b.
- 137Huth, J. R.; Mendoza, R.; Olejniczak, E. T.; Johnson, R. W.; Cothron, D. A.; Liu, Y.; Lerner, C. G.; Chen, J.; Hajduk, P. J. ALARM NMR: A Rapid and Robust Experimental Method to Detect Reactive False Positives in Biochemical Screens. J. Am. Chem. Soc. 2005, 127 (1), 217– 224, DOI: 10.1021/ja0455547[ACS Full Text], [CAS], Google Scholar137https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVCrurrF&md5=466332d4bdcddf6bc8a630aacbbb32efALARM NMR: a rapid and robust experimental method to detect reactive false positives in biochemical screensHuth, Jeffrey R.; Mendoza, Renaldo; Olejniczak, Edward T.; Johnson, Robert W.; Cothron, Darlene A.; Liu, Yaya; Lerner, Claude G.; Chen, Jun; Hajduk, Philip J.Journal of the American Chemical Society (2005), 127 (1), 217-224CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)High-throughput screening (HTS) of large compd. collections typically results in numerous small mol. hits that must be carefully evaluated to identify valid drug leads. Although several filtering mechanisms and other tools exist that can assist the chemist in this process, it is often the case that costly synthetic resources are expended in pursuing false positives. We report here a rapid and reliable NMR-based method for identifying reactive false positives including those that oxidize or alkylate a protein target. Importantly, the reactive species need not be the parent compd., as both reactive impurities and breakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactive mols. by NMR) and is based on monitoring DTT-dependent 13C chem. shift changes of the human La antigen in the presence of a test compd. or mixt. Extensive validation has been performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity. This included comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing a collection of more than 3500 compds. contg. HTS hits from 23 drug targets. The data show that current in silico filtering tools fail to identify more than half of the compds. that can act via reactive mechanisms. Significantly, we show how ALARM NMR data has been crit. in identifying reactive compds. that would otherwise have been prioritized for lead optimization. In addn., a new filtering tool has been developed on the basis of the ALARM NMR data that can augment current in silico programs for identifying nuisance compds. and improving the process of hit triage.
- 138Boelsterli, U. A.; Ho, H. K.; Zhou, S.; Leow, K. Y. Bioactivation and Hepatotoxicity of Nitroaromatic Drugs. Curr. Drug Metab. 2006, 7, 715– 727, DOI: 10.2174/138920006778520606[Crossref], [PubMed], [CAS], Google Scholar138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVWgt7rF&md5=21e155ba17d1ad097e7652a5d22728b3Bioactivation and hepatotoxicity of nitroaromatic drugsBoelsterli, Urs A.; Ho, Han Kiat; Zhou, Shufeng; Leow, Koon YeowCurrent Drug Metabolism (2006), 7 (7), 715-727CODEN: CDMUBU; ISSN:1389-2002. (Bentham Science Publishers Ltd.)A review. Certain drugs contg. a nitroarom. moiety (e.g., tolcapone, nimesulide, nilutamide, flutamide, nitrofurantoin) have been assocd. with organ-selective toxicity including rare cases of idiosyncratic liver injury. What they have in common is the potential for multistep nitroreductive bioactivation (6-electron transfer) that produces the potentially hazardous nitroanion radical, nitroso intermediate, and N-hydroxy deriv. These intermediates have been assocd. with increased oxidant stress and targeting of nucleophilic residues on proteins and nucleic acids. However, other mechanisms including the formation of oxidative metabolites and mitochondrial liability, as well as inherent toxicokinetic properties, also det. the drugs' overall potency. Therefore, structural modification not only of the nitro moiety but also of ring substituents can greatly reduced toxicity. Novel concepts have revealed that, besides the classical microsomal nitroreductases, cytosolic and mitochondrial enzymes including nitric oxide synthase can also bioactivate certain nitroarenes (nilutamide). Furthermore, animal models of silent mitochondrial dysfunction have demonstrated that a mitochondrial oxidant stress posed by certain nitroarom. drugs (nimesulide) can produce significant mitochondrial injury if superimposed on a genetic mitochondrial abnormality. Finally, there may be mechanisms for all nitroarom. drugs that do not involve bioactivation of the nitro group, e.g., AHR interactions with flutamide. Taken together, the focus of research on the hepatic toxicity of nitroarene-contg. drugs has shifted over the past years from the identification of the reactive intermediates generated during the bioreductive pathway to the underlying biomechanisms of liver injury. Most likely one of the next paradigm shifts will include the identification of determinants of susceptibility to nitroarom. drug-induced hepatotoxicity.
- 139Zeng, Q.; Nair, A. G.; Rosenblum, S. B.; Huang, H.-C.; Lesburg, C. A.; Jiang, Y.; Selyutin, O.; Chan, T.-Y.; Bennett, F.; Chen, K. X.; Venkatraman, S.; Sannigrahi, M.; Velazquez, F.; Duca, J. S.; Gavalas, S.; Huang, Y.; Pu, H.; Wang, L.; Pinto, P.; Vibulbhan, B.; Agrawal, S.; Ferrari, E.; Jiang, C.; Li, C.; Hesk, D.; Gesell, J.; Sorota, S.; Shih, N.-Y.; Njoroge, F. G.; Kozlowski, J. A. Discovery of an Irreversible HCV NS5B Polymerase Inhibitor. Bioorg. Med. Chem. Lett. 2013, 23 (24), 6585– 6587, DOI: 10.1016/j.bmcl.2013.10.060[Crossref], [PubMed], [CAS], Google Scholar139https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVWns7zK&md5=15a6315c05dd2c4c1c55420e317756ecDiscovery of an irreversible HCV NS5B polymerase inhibitorZeng, Qingbei; Nair, Anilkumar G.; Rosenblum, Stuart B.; Huang, Hsueh-Cheng; Lesburg, Charles A.; Jiang, Yueheng; Selyutin, Oleg; Chan, Tin-Yau; Bennett, Frank; Chen, Kevin X.; Venkatraman, Srikanth; Sannigrahi, Mousumi; Velazquez, Francisco; Duca, Jose S.; Gavalas, Stephen; Huang, Yuhua; Pu, Haiyan; Wang, Li; Pinto, Patrick; Vibulbhan, Bancha; Agrawal, Sony; Ferrari, Eric; Jiang, Chuan-kui; Li, Cheng; Hesk, David; Gesell, Jennifer; Sorota, Steve; Shih, Neng-Yang; Njoroge, F. George; Kozlowski, Joseph A.Bioorganic & Medicinal Chemistry Letters (2013), 23 (24), 6585-6587CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)The discovery of lead compd. 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray anal. The results of distribution, metab. and pharmacokinetics were reported. Compd. 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 μM), highly selective, and safe in in vitro and in vivo assays.
- 140Sato, K.; Kunitomo, Y.; Kasai, Y.; Utsumi, S.; Suetake, I.; Tajima, S.; Ichikawa, S.; Matsuda, A. Mechanism-Based Inhibitor of DNA Cytosine-5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2-Amino-4-Halopyridine-C-Nucleoside. ChemBioChem 2018, 19 (8), 865– 872, DOI: 10.1002/cbic.201700688[Crossref], [PubMed], [CAS], Google Scholar140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXktlyksLw%253D&md5=73265c9833d45b2931daf7ef0a251916Mechanism-Based Inhibitor of DNA Cytosine-5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2-Amino-4-Halopyridine-C-NucleosideSato, Kousuke; Kunitomo, Yuma; Kasai, Yukiko; Utsumi, Shohei; Suetake, Isao; Tajima, Shoji; Ichikawa, Satoshi; Matsuda, AkiraChemBioChem (2018), 19 (8), 865-872CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)In chromatin, 5-methylcytosine (mC), which represents the fifth nucleobase in genomic DNA, plays a role as an inducer of epigenetic changes. Tumor cells exhibit aberrant DNA methylation patterns, and inhibition of human DNA cytosine-5 methyltransferase (DNMT), which is responsible for generating mC in CpG sequences, is an effective strategy to treat various cancers. Here, we describe the design, synthesis, and evaluation of the properties of 2-amino-4-halopyridine-C-nucleosides (dXP) and oligodeoxyribonucleotides (ODNs) contg. dXP as a novel mechanism-based inhibitor of DNMTs. The designed ODN contg. XPpG forms a complex with DNMTs by covalent bonding through a nucleophilic arom. substitution (SNAr) reaction, and its cell proliferation activity is investigated. This study suggests that dXP in a CpG sequence of DNA could serve as a potential nucleic acid drug lead in cancer chemotherapy and a useful chem. probe for studies of epigenetics. Our mol. design using a SNAr reaction would be useful for DNMTs and other protein-DNA interactions.
- 141Kasai, Y.; Sato, K.; Utsumi, S.; Ichikawa, S. Improvement of SNAr Reaction Rate by an Electron-Withdrawing Group in the Crosslinking of DNA Cytosine-5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor. ChemBioChem 2018, 19 (17), 1866– 1872, DOI: 10.1002/cbic.201800244[Crossref], [PubMed], [CAS], Google Scholar141https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlWqurnN&md5=73faed58b2396d901a27c49b2aa13af3Improvement of SNAr Reaction Rate by an Electron-Withdrawing Group in the Crosslinking of DNA Cytosine-5 Methyltransferase by a Covalent Oligodeoxyribonucleotide InhibitorKasai, Yukiko; Sato, Kousuke; Utsumi, Shohei; Ichikawa, SatoshiChemBioChem (2018), 19 (17), 1866-1872CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)DNA cytosine 5-methyltransferase (DNMT) catalyzes methylation at the C5 position of the cytosine residues in the CpG sequence. Aberrant DNA methylation patterns are found in cancer cells. Therefore, inhibition of human DNMT is an effective strategy for treating various cancers. The inhibitors of DNMT have an electron-deficient nucleobase because this group facilitates attack by the catalytic Cys residue in DNMTs. Recently, we reported the synthesis and properties of mechanism-based modified nucleosides, 2-amino-4-halopyridine-C-nucleosides (dXP), as inhibitors of DNMT. To develop a more efficient inhibitor of DNMT for oligonucleotide therapeutics, oligodeoxyribonucleotides (ODNs) contg. other nucleoside analogs, which react more quickly with DNMT, are needed. Herein, we describe the design, synthesis, and evaluation of the properties of 2-amino-3-cyano-4-halopyridine-C-nucleosides (dXPCN) and ODNs contg. dXPCN, as more reactive inhibitors of DNMTs. Nucleophilic arom. substitution (SNAr) of the designed nucleosides, dXPCN, was faster than that of dXP, and the ODN contg. dXPCN effectively formed a complex with DNMTs. This study suggests that the incorporation of an electron-withdrawing group would be an effective method to increase reactivity toward the nucleophile of the DNMTs, while maintaining high specificity.
- 142Gianatassio, R.; Lopchuk, J. M.; Wang, J.; Pan, C.-M.; Malins, L. R.; Prieto, L.; Brandt, T. A.; Collins, M. R.; Gallego, G. M.; Sach, N. W.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Amination. Science 2016, 351 (6270), 241– 246, DOI: 10.1126/science.aad6252[Crossref], [PubMed], [CAS], Google Scholar142https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlvVCjuw%253D%253D&md5=868e44945c04c09881ac0d2516be23aeStrain-release aminationGianatassio, Ryan; Lopchuk, Justin M.; Wang, Jie; Pan, Chung-Mao; Malins, Lara R.; Prieto, Liher; Brandt, Thomas A.; Collins, Michael R.; Gallego, Gary M.; Sach, Neal W.; Spangler, Jillian E.; Zhu, Huichin; Zhu, Jinjiang; Baran, Phil S.Science (Washington, DC, United States) (2016), 351 (6270), 241-246CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)To optimize drug candidates, modern medicinal chemists are increasingly turning to an unconventional structural motif: small, strained ring systems. However, the difficulty of introducing substituents such as bicyclo[1.1.1]pentanes, azetidines, or cyclobutanes often outweighs the challenge of synthesizing the parent scaffold itself. Thus, there is an urgent need for general methods to rapidly and directly append such groups onto core scaffolds. Here we report a general strategy to harness the embedded potential energy of effectively spring-loaded C-C and C-N bonds with the most oft-encountered nucleophiles in pharmaceutical chem., amines. Strain-release amination can diversify a range of substrates with a multitude of desirable bioisosteres at both the early and late stages of a synthesis. The technique has also been applied to peptide labeling and bioconjugation.
- 143Lopchuk, J. M.; Fjelbye, K.; Kawamata, Y.; Malins, L. R.; Pan, C.-M.; Gianatassio, R.; Wang, J.; Prieto, L.; Bradow, J.; Brandt, T. A.; Collins, M. R.; Elleraas, J.; Ewanicki, J.; Farrell, W.; Fadeyi, O. O.; Gallego, G. M.; Mousseau, J. J.; Oliver, R.; Sach, N. W.; Smith, J. K.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Heteroatom Functionalization: Development, Scope, and Stereospecificity. J. Am. Chem. Soc. 2017, 139 (8), 3209– 3226, DOI: 10.1021/jacs.6b13229[ACS Full Text], [CAS], Google Scholar143https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVykur0%253D&md5=a7bd930dfd87427f5b8c64cb16fdbbe9Strain-Release Heteroatom Functionalization: Development, Scope, and StereospecificityLopchuk, Justin M.; Fjelbye, Kasper; Kawamata, Yu; Malins, Lara R.; Pan, Chung-Mao; Gianatassio, Ryan; Wang, Jie; Prieto, Liher; Bradow, James; Brandt, Thomas A.; Collins, Michael R.; Elleraas, Jeff; Ewanicki, Jason; Farrell, William; Fadeyi, Olugbeminiyi O.; Gallego, Gary M.; Mousseau, James J.; Oliver, Robert; Sach, Neal W.; Smith, Jason K.; Spangler, Jillian E.; Zhu, Huichin; Zhu, Jinjiang; Baran, Phil S.Journal of the American Chemical Society (2017), 139 (8), 3209-3226CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Driven by the ever-increasing pace of drug discovery and the need to push the boundaries of unexplored chem. space, medicinal chemists are routinely turning to unusual strained bioisosteres such as bicyclo[1.1.1]pentane, azetidine, and cyclobutane to modify their lead compds. Too often, however, the difficulty of installing these fragments surpasses the challenges posed even by the construction of the parent drug scaffold. This full account describes the development and application of a general strategy where spring-loaded, strained C-C and C-N bonds react with amines to allow for the "any-stage" installation of small, strained ring systems. In addn. to the functionalization of small building blocks and late-stage intermediates, the methodol. has been applied to bioconjugation and peptide labeling. For the first time, the stereospecific strain-release "cyclopentylation" of amines, alcs., thiols, carboxylic acids, and other heteroatoms is introduced. This report describes the development, synthesis, scope of reaction, bioconjugation, and synthetic comparisons of four new chiral "cyclopentylation" reagents.
- 144Semmler, K.; Szeimies, G.; Belzner, J. Tetracyclo[5.1.0.01,6.02,7]Octane, a [1.1.1]Propellane Derivative, and a New Route to the Parent Hydrocarbon. J. Am. Chem. Soc. 1985, 107 (22), 6410– 6411, DOI: 10.1021/ja00308a053[ACS Full Text], [CAS], Google Scholar144https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlslOqurc%253D&md5=a3846ab7c1f01f056f62e0a5e42a0076Tetracyclo[5.1.0.01,6.02,7]octane, a [1.1.1]propellane derivative, and a new route to the parent hydrocarbonSemmler, Klaus; Szeimies, Guenter; Belzner, JohannesJournal of the American Chemical Society (1985), 107 (22), 6410-11CODEN: JACSAT; ISSN:0002-7863.The title compd. (I) was prepd. from tricyclo[4.1.0.02,7]heptane. Carbenation (CHBr3) of (ClCH2)2C:CBr2, then cyclization with BuLi gave propellane (II).
- 145Haag, B.; Mosrin, M.; Ila, H.; Malakhov, V.; Knochel, P. Regio- and Chemoselective Metalation of Arenes and Heteroarenes Using Hindered Metal Amide Bases. Angew. Chem., Int. Ed. 2011, 50 (42), 9794– 9824, DOI: 10.1002/anie.201101960[Crossref], [CAS], Google Scholar145https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1Cjs7jI&md5=d8ed72b4eb7c627230c658547eb030f4Regio- and Chemoselective Metalation of Arenes and Heteroarenes Using Hindered Metal Amide BasesHaag, Benjamin; Mosrin, Marc; Ila, Hiriyakkanavar; Malakhov, Vladimir; Knochel, PaulAngewandte Chemie, International Edition (2011), 50 (42), 9794-9824CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The prepn. of highly functionalized organometallic compds. can be achieved by direct C-H activation of a broad range of unsatd. substrates using lithium chloride solubilized 2,2,6,6-tetramethylpiperidide bases (TMPnMXmp LiCl). These are excellent reagents for converting a wide range of arom. and heterocyclic substrates into valuable organometallic reagents with broad applications in org. synthesis.
- 146Wishart, D. S.; Feunang, Y. D.; Guo, A. C.; Lo, E. J.; Marcu, A.; Grant, J. R.; Sajed, T.; Johnson, D.; Li, C.; Sayeeda, Z.; Assempour, N.; Iynkkaran, I.; Liu, Y.; Maciejewski, A.; Gale, N.; Wilson, A.; Chin, L.; Cummings, R.; Le, D.; Pon, A.; Knox, C.; Wilson, M. DrugBank 5.0: A Major Update to the DrugBank Database for 2018. Nucleic Acids Res. 2018, 46 (D1), D1074– D1082, DOI: 10.1093/nar/gkx1037[Crossref], [PubMed], [CAS], Google Scholar146https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlGisbvI&md5=986b28c7ea546596a26dd3ba38f05feeDrugBank 5.0: a major update to the DrugBank database for 2018Wishart, David S.; Feunang, Yannick D.; Guo, An C.; Lo, Elvis J.; Marcu, Ana; Grant, Jason R.; Sajed, Tanvir; Johnson, Daniel; Li, Carin; Sayeeda, Zinat; Assempour, Nazanin; Iynkkaran, Ithayavani; Liu, Yifeng; Maciejewski, Adam; Gale, Nicola; Wilson, Alex; Chin, Lucy; Cummings, Ryan; Le, Diana; Pon, Allison; Knox, Craig; Wilson, MichaelNucleic Acids Research (2018), 46 (D1), D1074-D1082CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)DrugBank is a web-enabled database contg. comprehensivemol. information about drugs, their mechanisms, their interactions and their targets. First described in 2006, Drug- Bank has continued to evolve over the past 12 years in response to marked improvements to web stds. and changing needs for drug research and development. This year's update, DrugBank 5.0, represents the most significant upgrade to the database in more than 10 years. In many cases, existing data content has grown by 100% or more over the last update. For instance, the total no. of investigational drugs in the database has grown by almost 300%, the no. of drug-drug interactions has grown by nearly 600% and the no. of SNP-assocd. drug effects has grown more than 3000%. Significant improvements have been made to the quantity, quality and consistency of drug indications, drug binding data as well as drug-drug and drug-food interactions. A great deal of brand new data have also been added to DrugBank 5.0. This includes information on the influence of hundreds of drugs on metabolite levels (pharmacometabolomics), gene expression levels (pharmacotranscriptomics) and protein expression levels (pharmacoproteomics). New data have also been added on the status of hundreds of newdrug clin. trials and existing drug repurposing trials. Many other important improvements in the content, interface and performance of the DrugBank website have been made and these should greatly enhance its ease of use, utility and potential applications in many areas of pharmacol. research, pharmaceutical science and drug education.
- 147Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Applications of Fluorine in Medicinal Chemistry. J. Med. Chem. 2015, 58 (21), 8315– 8359, DOI: 10.1021/acs.jmedchem.5b00258[ACS Full Text], [CAS], Google Scholar147https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1ajs7%252FK&md5=9995829a94a8c0b8d9fb0d21bdfd5a1dApplications of Fluorine in Medicinal ChemistryGillis, Eric P.; Eastman, Kyle J.; Hill, Matthew D.; Donnelly, David J.; Meanwell, Nicholas A.Journal of Medicinal Chemistry (2015), 58 (21), 8315-8359CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review with meta-anal. The role of fluorine in drug design and development is expanding rapidly as we learn more about the unique properties assocd. with this unusual element and how to deploy it with greater sophistication. The judicious introduction of fluorine into a mol. can productively influence conformation, pKa, intrinsic potency, membrane permeability, metabolic pathways, and pharmacokinetic properties. In addn., 18F has been established as a useful positron emitting isotope for use with in vivo imaging technol. that potentially has extensive application in drug discovery and development, often limited only by convenient synthetic accessibility to labeled compds. The wide ranging applications of fluorine in drug design are providing a strong stimulus for the development of new synthetic methodologies that allow more facile access to a wide range of fluorinated compds. In this review, we provide an update on the effects of the strategic incorporation of fluorine in drug mols. and applications in positron emission tomog.
- 148Meanwell, N. A. Fluorine and Fluorinated Motifs in the Design and Application of Bioisosteres for Drug Design. J. Med. Chem. 2018, 61 (14), 5822– 5880, DOI: 10.1021/acs.jmedchem.7b01788[ACS Full Text], [CAS], Google Scholar148https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2qu7w%253D&md5=2d0ce3326c7ff932da8d7d26972ced14Fluorine and Fluorinated Motifs in the Design and Application of Bioisosteres for Drug DesignMeanwell, Nicholas A.Journal of Medicinal Chemistry (2018), 61 (14), 5822-5880CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The electronic properties and relatively small size of fluorine endow it with considerable versatility as a bioisostere and it has found application as a substitute for lone pairs of electrons, the hydrogen atom, and the Me group while also acting as a functional mimetic of the carbonyl, carbinol, andnitrile moieties. In this context, fluorine substitution can influence the potency, conformation, metab., membrane permeability, and P-gp recognition of a mol. and temper inhibition of the hERG channel by basic amines. However, as a consequence of the unique properties of fluorine, it features prominently in the design of higher order structural metaphors that are more esoteric in their conception and which reflect a more sophisticated mol. construction that broadens biol. mimesis. In this Perspective, applications of fluorine in the construction of bioisosteric elements designed to enhance the in vitro and in vivo properties of a mol. are summarized.
- 149Grishin, N. V.; Osterman, A. L.; Brooks, H. B.; Phillips, M. A.; Goldsmith, E. J. X-Ray Structure of Ornithine Decarboxylase from Trypanosoma Brucei: The Native Structure and the Structure in Complex with α-Difluoromethylornithine. Biochemistry 1999, 38 (46), 15174– 15184, DOI: 10.1021/bi9915115[ACS Full Text], [CAS], Google Scholar149https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXmvFCrurs%253D&md5=83ae6756feca1823788b041bbf331542X-ray structure of ornithine decarboxylase from Trypanosoma brucei: The native structure and the structure in complex with α-difluoromethylornithineGrishin, Nick V.; Osterman, Andrei L.; Brooks, Harold B.; Phillips, Margaret A.; Goldsmith, Elizabeth J.Biochemistry (1999), 38 (46), 15174-15184CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by T. brucei. One of the currently used drugs, α-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO was detd. by x-ray crystallog. to 2.0 Å resoln. The protein crystd. in space group P21 (a = 66.8, b = 154.5, c = 77.1 Å, and β = 90.58°), with 2 dimers per asym. unit. The initial phasing was done by mol. replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also detd. to 2.9 Å resoln. by mol. replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC was a β/α-barrel, and the C-terminal domain of ODC was a modified Greek key β-barrel. In comparison to structurally related alanine racemase, the 2 domains were rotated 27° relative to each other. In addn., 2 of the β-strands in the C-terminal domain had exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface were formed primarily by the C-terminal domains, which interacted through 6 arom. rings that formed stacking interactions across the domain boundary. The PLP-binding site was formed by the C-termini of β-strands and loops in the β/α-barrel. In the native structure, Lys-69 formed a Schiff base with PLP. In both structures, the phosphate of PLP was bound between the 7th and 8th strands forming interactions with Arg-277 and a Gly loop (residues 235-237). The pyridine N atom of PLP interacted with Glu-274. DFMO formed a Schiff base with PLP and was covalently attached to Cys-360. It was bound at the dimer interface and the δ-C amino group of DFMO was positioned between Asp-361 of one subunit and Asp-332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 had rotated 145° toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes were obsd. upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.
- 150Eckstein, J. W.; Foster, P. G.; Finer-Moore, J.; Wataya, Y.; Santi, D. V. Mechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2’-Deoxyuridine 5′-Monophosphate. Biochemistry 1994, 33 (50), 15086– 15094, DOI: 10.1021/bi00254a018[ACS Full Text], [CAS], Google Scholar150https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmvFGrt70%253D&md5=f82c290ac5cb6d16115a3517f283477aMechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2'-deoxyuridine 5'-MonophosphateEckstein, Jens W.; Foster, Paul G.; Finer-Moore, Janet; Wataya, Yusuke; Santi, Daniel V.Biochemistry (1994), 33 (50), 15086-94CODEN: BICHAW; ISSN:0006-2960.Thymidylate synthase (TS) from Lactobacillus casei is inhibited by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF3dUMP). CF3dUMP binds to the active site of TS in the absence of CH2H4folate, and attack of the catalytic nucleophile cysteine 198 at C6 of the pyrimidine leads to activation of the trifluoromethyl group and release of fluoride ion. Subsequently, the activated heterocycle reacts with a nucleophile of the enzyme to form a moderately stable covalent complex. Proteolytic digestion of TS treated with [2'-3H]CF3dUMP, followed by sequencing of the labeled peptides, revealed that tyrosine 146 and cysteine 198 are covalently bound to the inhibitor in the enzyme-inhibitor complex. The presence of dithiothreitol (DTT) or β-mercaptoethanol resulted in the breakdown of the covalent complex, and products from the breakdown of the complex were isolated and characterized. The three-dimensional structure of the enzyme-inhibitor complex was detd. by x-ray crystallog., clearly demonstrating covalent attachment of the nucleotide to tyrosine 146. A chem. reaction mechanism for the inhibition of TS by CF3dUMP is presented that is consistent with the kinetic, biochem., and structural results.
- 151Cohen, M. S.; Zhang, C.; Shokat, K. M.; Taunton, J. Structural Bioinformatics-Based Design of Selective, Irreversible Kinase Inhibitors. Science 2005, 308 (5726), 1318– 1321, DOI: 10.1126/science1108367[Crossref], [PubMed], [CAS], Google Scholar151https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXks1Ciurs%253D&md5=1364444dada4d1d46510aa0212428952Structural Bioinformatics-Based Design of Selective, Irreversible Kinase InhibitorsCohen, Michael S.; Zhang, Chao; Shokat, Kevan M.; Taunton, JackScience (Washington, DC, United States) (2005), 308 (5726), 1318-1321CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The active sites of 491 human protein kinase domains are highly conserved, which makes the design of selective inhibitors a formidable challenge. We used a structural bioinformatics approach to identify two selectivity filters, a threonine and a cysteine, at defined positions in the active site of p90 ribosomal protein S6 kinase (RSK). A fluoromethylketone inhibitor, designed to exploit both selectivity filters, potently and selectively inactivated RSK1 and RSK2 in mammalian cells. Kinases with only one selectivity filter were resistant to the inhibitor, yet they became sensitized after genetic introduction of the second selectivity filter. Thus, two amino acids that distinguish RSK from other protein kinases are sufficient to confer inhibitor sensitivity.
- 152Cohen, M. S.; Hadjivassiliou, H.; Taunton, J. A Clickable Inhibitor Reveals Context-Dependent Autoactivation of P90 RSK. Nat. Chem. Biol. 2007, 3 (3), 156– 160, DOI: 10.1038/nchembio859[Crossref], [PubMed], [CAS], Google Scholar152https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhslWgsL4%253D&md5=68f7da1c274678e884ea71d1078b092cA clickable inhibitor reveals context-dependent autoactivation of p90 RSKCohen, Michael S.; Hadjivassiliou, Haralambos; Taunton, JackNature Chemical Biology (2007), 3 (3), 156-160CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)P90 ribosomal protein S6 kinases (RSKs) integrate upstream signals through two catalytic domains. Autophosphorylation of Ser386 by the regulatory C-terminal kinase domain (CTD) is thought to be essential for activation of the N-terminal kinase domain (NTD), which phosphorylates multiple downstream targets. We recently reported fmk, an irreversible inhibitor of the CTD of RSK1 and RSK2. Here we describe fmk-pa, a propargylamine variant that has improved cellular potency and a 'clickable' tag for assessing the extent and selectivity of covalent RSK modification. Copper-catalyzed conjugation of an azidoalkyl reporter (the click reaction) revealed that fmk-pa achieves selective and saturable modification of endogenous RSK1 and RSK2 in mammalian cells. Satg. concns. of fmk-pa inhibited Ser386 phosphorylation and downstream signaling in response to phorbol ester stimulation, but had no effect on RSK activation by lipopolysaccharide. RSK autoactivation by the CTD is therefore context dependent, which suggests that NTD and CTD inhibitors should have distinct physiol. effects.
- 153Carroll, F. A. Perspectives on Structure and Mechanism in Organic Chemistry, 2nd ed.; Wiley-VCH: Weinheim, 2014.
- 154Shaik, S. S. The .Alpha.- and .Beta.-Carbon Substituent Effect on SN2 Reactivity. A Valence-Bond Approach. J. Am. Chem. Soc. 1983, 105 (13), 4359– 4367, DOI: 10.1021/ja00351a039[ACS Full Text], [CAS], Google Scholar154https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXkt1WlsrY%253D&md5=36aa6ec687f8a91cbf37372449cfaf05The α- and β-carbon substituent effect on SN2 reactivity. A valence-bond approachShaik, Sason S.Journal of the American Chemical Society (1983), 105 (13), 4359-67CODEN: JACSAT; ISSN:0002-7863.α- And β-C substituent effect, on SN2 reactivity and reactivity-selectivity, is discussed by using a (previously described) correlation diagram model of the SN2 reaction. The reaction barrier (E) is a fraction (f) of the energy gap (IN: - ARX) between the 2 curves which intersect to yield the reaction profile. IN: Is the ionization potential of the nucleophile (N:) and ARX is the electron affinity of the substrate (RX). The f which enters the activation barrier depends inter alia on the degree of delocalization of the three-electron bonds, e.g., (R···X)-. The more delocalized the three-electron bond, the larger the f. Reactivity trends arise from the interplay between the electron transfer aspect (IN: - ARX) and the bond-interchange aspect (e.g., the degree of delocalization of the three-electron bonds) of the SN2 transformation. α-Halo substitution (on R) delocalizes the three-electron bond and effects a small improvement in the acceptor ability of the substrate; it therefore slows down SN2 reactivity. The largest delocalization is effected when the α-substituent(s) is (are) identical with the leaving group. In these cases, the strongest rate retardation is obsd. π-Acceptor α-substituents improve the substrate acceptor ability markedly without greatly delocalizing the three-electron bonds. Therefore, these substituents will enhance reactivity but mainly toward powerful nucleophiles. The effects of other α-substituents (e.g., MeO, Ph, SiR3, etc.) and β-substituents (e.g., F, Cl, Br, RO, etc.) are also discussed. The reactivity reversals often reported in the literature are suggested to be manifestations of the gap-slope interplay. The reactivity of alkyl halides is extensively examd.
- 155Lundell, N.; Schreitmüller, T. Sample Preparation for Peptide Mapping— A Pharmaceutical Quality-Control Perspective. Anal. Biochem. 1999, 266 (1), 31– 47, DOI: 10.1006/abio.1998.2919[Crossref], [PubMed], [CAS], Google Scholar155https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXisV2ltQ%253D%253D&md5=0d20ad326def5e9bdf5df9d1e7ee1961Sample preparation for peptide mapping - a pharmaceutical quality-control perspectiveLundell, Niklas; Schreitmuller, ThomasAnalytical Biochemistry (1999), 266 (1), 31-47CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)A review with 116 refs. In quality control of therapeutic proteins peptide mapping is used for confirmation of primary structure and detection of posttranslational modifications. The demands put on the exptl. procedure are therefore different than in the case of detn. of an unknown protein structure. It is here recognized that a peptide-mapping method for quality control of proteins should be inert (not induce or revert modifications), general, robust, and allow a high sample throughput. The steps prior to the sepn. of the generated peptides are identified as crucial for meeting these demands. This includes denaturation, redn., alkylation, buffer exchange, solubilization, and digestion. A crit. review of the literature regarding these steps is presented. Relevant options in all steps are exptl. evaluated. Novel approaches are developed for many of the steps. The result is a sample prepn. procedure that essentially meets the stated demands. (c) 1999 Academic Press.
- 156Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B. D.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F. Quantitative Reactivity Profiling Predicts Functional Cysteines in Proteomes. Nature 2010, 468 (7325), 790– 795, DOI: 10.1038/nature09472[Crossref], [PubMed], [CAS], Google Scholar156https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVGhsr%252FI&md5=277e436e7a8f4dfff0b548bce52ac40dQuantitative reactivity profiling predicts functional cysteines in proteomesWeerapana, Eranthie; Wang, Chu; Simon, Gabriel M.; Richter, Florian; Khare, Sagar; Dillon, Myles B. D.; Bachovchin, Daniel A.; Mowen, Kerri; Baker, David; Cravatt, Benjamin F.Nature (London, United Kingdom) (2010), 468 (7325), 790-795CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochem. functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quant. the intrinsic reactivity of cysteine residues en masse directly in native biol. systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulfur protein biogenesis. We also demonstrate that quant. reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.
- 157Weerapana, E.; Simon, G. M.; Cravatt, B. F. Disparate Proteome Reactivity Profiles of Carbon Electrophiles. Nat. Chem. Biol. 2008, 4 (7), 405– 407, DOI: 10.1038/nchembio.91[Crossref], [PubMed], [CAS], Google Scholar157https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnt1ejtL8%253D&md5=69eb0ae589c50f46a8695dc6731366e2Disparate proteome reactivity profiles of carbon electrophilesWeerapana, Eranthie; Simon, Gabriel M.; Cravatt, Benjamin F.Nature Chemical Biology (2008), 4 (7), 405-407CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Insights into the proteome reactivity of electrophiles are crucial for designing activity-based probes for enzymes lacking cognate affinity labels. Here, the authors show that different classes of carbon electrophiles exhibit markedly distinct amino acid labeling profiles in proteomes, ranging from selective reactivity with cysteine to adducts with several amino acids. These data specify electrophilic chemotypes with restricted and permissive reactivity profiles to guide the design of next-generation functional proteomics probes.
- 158Lonsdale, R.; Burgess, J.; Colclough, N.; Davies, N. L.; Lenz, E. M.; Orton, A. L.; Ward, R. A. Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity. J. Chem. Inf. Model. 2017, 57 (12), 3124– 3137, DOI: 10.1021/acs.jcim.7b00553[ACS Full Text], [CAS], Google Scholar158https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsl2gsLjF&md5=722e83699c58dee1009b87c8c31bdeccExpanding the Armory: Predicting and Tuning Covalent Warhead ReactivityLonsdale, Richard; Burgess, Jonathan; Colclough, Nicola; Davies, Nichola L.; Lenz, Eva M.; Orton, Alexandra L.; Ward, Richard A.Journal of Chemical Information and Modeling (2017), 57 (12), 3124-3137CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)Targeted covalent inhibition is an established approach for increasing the potency and selectivity of potential drug candidates, as well as identifying potent and selective tool compds. for target validation studies. It is evident that identification of reversible recognition elements is essential for selective covalent inhibition, but this must also be achieved with the appropriate level of inherent reactivity of the reactive functionality (or "warhead"). Structural changes that increase or decrease warhead reactivity, guided by methods to predict the effect of those changes, have the potential to tune warhead reactivity and negate issues related to potency and/or toxicity. The half-life to adduct formation with glutathione (GSH t1/2) is a useful assay for measuring the reactivity of cysteine-targeting covalent warheads but is limited to synthesized mols. In this manuscript the authors assess the ability of several exptl. and computational approaches to predict GSH t1/2 for a range of cysteine targeting warheads, including a novel method based on pKa. Furthermore, matched mol. pairs anal. has been performed against the internal compd. collection, revealing structure-activity relationships between a selection of different covalent warheads. These observations and methods of prediction will be valuable in the design of new covalent inhibitors with desired levels of reactivity.
- 159Allimuthu, D.; Adams, D. J. 2-Chloropropionamide As a Low-Reactivity Electrophile for Irreversible Small-Molecule Probe Identification. ACS Chem. Biol. 2017, 12 (8), 2124– 2131, DOI: 10.1021/acschembio.7b00424[ACS Full Text], [CAS], Google Scholar159https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpvVGqsrw%253D&md5=39b4794ebb55d3314a7bac5b04ddadc72-Chloropropionamide As a Low-Reactivity Electrophile for Irreversible Small-Molecule Probe IdentificationAllimuthu, Dharmaraja; Adams, Drew J.ACS Chemical Biology (2017), 12 (8), 2124-2131CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Resurgent interest in covalent target engagement in drug discovery has demonstrated that small mols. contg. weakly reactive electrophiles can be safe and effective therapies. Several recently FDA-approved drugs feature an acrylamide functionality to selectively engage cysteine side chains of kinases (Ibrutinib, Afatinib, and Neratinib). Addnl. electrophilic functionalities whose reactivity is compatible with highly selective target engagement and in vivo application could open new avenues in covalent small mol. discovery. Here the authors report the synthesis and evaluation of a library of small mols. contg. the 2-chloropropionamide functionality, which the authors demonstrate is less reactive than typical acrylamide electrophiles. Although many library members do not appear to label proteins in cells, the authors identified S-CW3554 as selectively labeling protein disulfide isomerase and inhibiting its enzymic activity. Subsequent profiling of the library against five diverse cancer cell lines showed unique cytotoxicity for S-CW3554 I in cells derived from multiple myeloma, a cancer recently reported to be sensitive to PDI inhibition. The novel PDI inhibitor highlights the potential of 2-chloropropionamides as weak and stereochem.-tunable electrophiles for covalent drug discovery.
- 160Steinmetz, C. G.; Xie, P.; Weiner, H.; Hurley, T. D. Structure of Mitochondrial Aldehyde Dehydrogenase: The Genetic Component of Ethanol Aversion. Structure 1997, 5 (5), 701– 711, DOI: 10.1016/S0969-2126(97)00224-4[Crossref], [PubMed], [CAS], Google Scholar160https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjvFamtLw%253D&md5=c66dc959c97a17f68490b588d9d5419aStructure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversionSteinmetz, Curtis G.; Xie, Peiguang; Weiner, Henry; Hurley, Thomas D.Structure (London) (1997), 5 (5), 701-711CODEN: STRUE6; ISSN:0969-2126. (Current Biology)The single genetic factor most strongly correlated with reduced alc. consumption and incidence of alcoholism is a naturally occurring variant of mitochondrial aldehyde dehydrogenase (ALDH2). This variant contains a glutamate to lysine substitution at position 487 (E487K). The E487K variant of ALDH2 is found in approx. 50% of the Asian population, and is assocd. with a phenotypic loss of ALDH2 activity in both heterozygotes and homozygotes. ALDH2-deficient individuals exhibit an averse response to ethanol consumption, which is probably caused by elevated levels of blood acetaldehyde. The structure of ALDH2 is important for the elucidation of its catalytic mechanism, to gain a clear understanding of the contribution of ALDH2 to the genetic component of alcoholism and for the development of specific ALDH2 inhibitors as potential drugs for use in the treatment of alcoholism. The x-ray structure of bovine ALDH2 has been solved to 2.65 Å in its free form and to 2.75 Å in a complex with NAD+. The enzyme structure contains three domains: two dinucleotide-binding domains and a small three-stranded β-sheet domain, which is involved in subunit interactions in this tetrameric enzyme. The E487K mutation occurs in this small oligomerization domain and is located at a key interface between subunits immediately below the active site of another monomer. The active site of ALDH2 is divided into two halves by the nicotinamide ring of NAD+. Adjacent to the A-side (Pro-R) of the nicotinamide ring is a cluster of three cysteines (Cys301, Cys302 and Cys303) and adjacent to the B-side (Pro-S) are Thr244, Glu268, Glu476 and an ordered water mol. bound to Thr244 and Glu476. Although there is a recognizable Rossmann-type fold, the coenzyme-binding region of ALDH2 binds NAD+ in a manner not seen in other NAD+-binding enzymes. The positions of the residues near the nicotinamide ring of NAD+ suggest a chem. mechanism whereby Glu268 functions as a general base through a bound water mol. The sidechain amide nitrogen of Asn169 and the peptide nitrogen of Cys302 are in position to stabilize the oxyanion present in the tetrahedral transition state prior to hydride transfer. The functional importance of residue Glu487 now appears to be due to indirect interactions of this residue with the substrate-binding site via Arg264 and Arg475.
- 161Karala, A.-R.; Lappi, A.-K.; Ruddock, L. W. Modulation of an Active-Site Cysteine PKa Allows PDI to Act as a Catalyst of Both Disulfide Bond Formation and Isomerization. J. Mol. Biol. 2010, 396 (4), 883– 892, DOI: 10.1016/j.jmb.2009.12.014[Crossref], [PubMed], [CAS], Google Scholar161https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitValtr4%253D&md5=7fb917968538e6ed0f15277e254ef224Modulation of an Active-Site Cysteine pKa Allows PDI to Act as a Catalyst of both Disulfide Bond Formation and IsomerizationKarala, Anna-Riikka; Lappi, Anna-Kaisa; Ruddock, Lloyd W.Journal of Molecular Biology (2010), 396 (4), 883-892CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Protein disulfide isomerase (PDI) plays a central role in disulfide bond formation in the endoplasmic reticulum. It is implicated both in disulfide bond formation and in disulfide bond redn. and isomerization. To be an efficient catalyst of all three reactions requires complex mechanisms. These include mechanisms to modulate the pKa values of the active-site cysteines of PDI. Here, the role of arginine 120 in modulating the pKa values of these cysteines was examd. It was found that arginine 120 plays a significant role in modulating the pKa of the C-terminal active-site cysteine in the a domain of PDI and plays a role in detg. the reactivity of the N-terminal active-site cysteine but not via direct modulation of its pKa. Mutation of arginine 120 and the corresponding residue, arginine 461, in the a' domain severely reduces the ability of PDI to catalyze disulfide bond formation and redn. but enhances the ability to catalyze disulfide bond isomerization due to the formation of more stable PDI-substrate mixed disulfides. These results suggest that the modulation of pKa of the C-terminal active cysteine by the movement of the side chain of these arginine residues into the active-site locales has evolved to allow PDI to efficiently catalyze both oxidn. and isomerization reactions.
- 162Wang, C.; Abegg, D.; Hoch, D. G.; Adibekian, A. Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT. Angew. Chem., Int. Ed. 2016, 55 (8), 2911– 2915, DOI: 10.1002/anie.201511301[Crossref], [CAS], Google Scholar162https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1Ojt74%253D&md5=37ddd0a42f0d2c87207fd8c27f3f9496Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMTWang, Chao; Abegg, Daniel; Hoch, Dominic G.; Adibekian, AlexanderAngewandte Chemie, International Edition (2016), 55 (8), 2911-2915CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors present a novel chem. scaffold for cysteine-reactive covalent inhibitors. Chloromethyl triazoles (CMTs) are readily accessed in only two chem. steps, thus enabling the rapid optimization of the pharmacol. properties of these inhibitors. The authors demonstrate the tunability of the CMTs towards a specific biol. target by synthesizing I as the first potent nonpseudosubstrate inhibitor of the O6-alkylguanine DNA methyltransferase (MGMT), a protein of major clin. significance for the treatment of several severe cancer forms. Using quant. proteomics profiling techniques, the authors show that I exhibits a high degree of selectivity towards MGMT. Finally, the authors validate the effectiveness of the MGMT inhibitor in combination with the DNA alkylating drug temozolomide in breast and colon cancer cells by fluorescence imaging and a cell-viability assay. The results may open a new avenue towards the development of a clin. approved MGMT inhibitor.
- 163Boren, B. C.; Narayan, S.; Rasmussen, L. K.; Zhang, L.; Zhao, H.; Lin, Z.; Jia, G.; Fokin, V. V. Ruthenium-Catalyzed Azide–Alkyne Cycloaddition: Scope and Mechanism. J. Am. Chem. Soc. 2008, 130 (28), 8923– 8930, DOI: 10.1021/ja0749993[ACS Full Text], [CAS], Google Scholar163https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXns1Wjtr8%253D&md5=8b5ba21060ad9d6984bba3e2587aa44dRuthenium-Catalyzed Azide-Alkyne Cycloaddition: Scope and MechanismBoren, Brant C.; Narayan, Sridhar; Rasmussen, Lars K.; Zhang, Li; Zhao, Haitao; Lin, Zhenyang; Jia, Guochen; Fokin, Valery V.Journal of the American Chemical Society (2008), 130 (28), 8923-8930CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The catalytic activity of ruthenium(II) complexes in azide-alkyne cycloaddns. were evaluated. The [Cp*RuCl] complexes, such as Cp*RuCl(PPh3)2, Cp*RuCl(COD), and Cp*RuCl(NBD), were among the most effective catalysts. In the presence of catalytic Cp*RuCl(PPh3)2 or Cp*RuCl(COD), primary and secondary azides react with a broad range of terminal alkynes contg. a range of functionalities selectively producing 1,5-disubstituted 1,2,3-triazoles; tertiary azides were significantly less reactive. Both complexes also promote the cycloaddn. reactions of org. azides with internal alkynes, providing access to fully-substituted 1,2,3-triazoles. The ruthenium-catalyzed azide-alkyne cycloaddn. (RuAAC) appears to proceed via oxidative coupling of the azide and alkyne reactants to give a six-membered ruthenacycle intermediate, in which the first new carbon-nitrogen bond is formed between the more electroneg. carbon of the alkyne and the terminal, electrophilic nitrogen of the azide. This step is followed by reductive elimination, which forms the triazole product. DFT calcns. support this mechanistic proposal and indicate that the reductive elimination step is rate-detg.
- 164Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Epoxide Electrophiles as Activity-Dependent Cysteine Protease Profiling and Discovery Tools. Chem. Biol. 2000, 7 (8), 569– 581, DOI: 10.1016/S1074-5521(00)00014-4[Crossref], [PubMed], [CAS], Google Scholar164https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnt1WnsbY%253D&md5=4bfc0e299524198ec324c96c8981baf7Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery toolsGreenbaum, Doron; Medzihradszky, Katalin F.; Burlingame, Alma; Bogyo, MatthewChemistry & Biology (2000), 7 (8), 569-581CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Science Ltd.)Background: Anal. of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly detg. activities of an entire class of enzymes on a genome-wide scale would be of great utility. Results: We have engineered chem. probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes contg. a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivs. was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular exts. Finally, the affinity tag of DCG-04 allowed purifn. of modified proteases and identification by mass spectrometry. Conclusions: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixts. These probes were used to det. relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purifn./identification of targets.
- 165Willems, L. I.; Jiang, J.; Li, K.-Y.; Witte, M. D.; Kallemeijn, W. W.; Beenakker, T. J. N.; Schröder, S. P.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. From Covalent Glycosidase Inhibitors to Activity-Based Glycosidase Probes. Chem. - Eur. J. 2014, 20 (35), 10864– 10872, DOI: 10.1002/chem.201404014[Crossref], [PubMed], [CAS], Google Scholar165https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlWmtLjO&md5=45059a66dcfd0a2895da38996d0a7be1From Covalent Glycosidase Inhibitors to Activity-Based Glycosidase ProbesWillems, Lianne I.; Jiang, Jianbing; Li, Kah-Yee; Witte, Martin D.; Kallemeijn, Wouter W.; Beenakker, Thomas J. N.; Schroeder, Sybrin P.; Aerts, Johannes M. F. G.; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; Overkleeft, Hermen S.Chemistry - A European Journal (2014), 20 (35), 10864-10872CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Activity-based protein profiling has emerged as a powerful discovery tool in chem. biol. and medicinal chem. research. Success of activity-based protein profiling hinges on the presence of compds. that can covalently and irreversibly bind to enzymes, do so selectively in the context of complex biol. samples, and subsequently report on the selected pool of proteins. Such tagged mols. featuring an electrophilic trap, termed activity-based probes, have been developed with most success for serine hydrolases and various protease families (serine proteases, cysteine proteases, proteasomes). This concept presents the current progress and future directions in the design of activity-based probes targeting retaining glycosidases, enzymes that employ a double displacement mechanism in the hydrolysis of glycosidic bonds with overall retention. In contrast to inverting glycosidases, retaining glycosidases form a covalent intermediate with their substrates during the catalytic process and are therefore amenable to activity-based protein profiling studies.
- 166Adams, B. T.; Niccoli, S.; Chowdhury, M. A.; Esarik, A. N. K.; Lees, S. J.; Rempel, B. P.; Phenix, C. P. N -Alkylated Aziridines Are Easily-Prepared, Potent, Specific and Cell-Permeable Covalent Inhibitors of Human β-Glucocerebrosidase. Chem. Commun. 2015, 51 (57), 11390– 11393, DOI: 10.1039/C5CC03828F[Crossref], [PubMed], [CAS], Google Scholar166https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVCmu7nI&md5=c5f088dbe0d16ac976f5070113e6f74bN-Alkylated aziridines are easily-prepared, potent, specific and cell-permeable covalent inhibitors of human β-glucocerebrosidaseAdams, B. T.; Niccoli, S.; Chowdhury, M. A.; Esarik, A. N. K.; Lees, S. J.; Rempel, B. P.; Phenix, C. P.Chemical Communications (Cambridge, United Kingdom) (2015), 51 (57), 11390-11393CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)β-Glucocerebrosidase deficiency leads to Gaucher disease and is a potential marker of Parkinson's disease. The authors have identified N-octyl conduritol aziridine as a potent and specific covalent inactivator of GBA1 in living cells. This compd. is a promising lead towards a positron emission tomog. probe intended to image GBA1 activity.
- 167Singh, G. S. Synthetic Aziridines in Medicinal Chemistry: A Mini-Review. Mini-Rev. Med. Chem. 2016, 16, 892– 904, DOI: 10.2174/1389557515666150709122244[Crossref], [PubMed], [CAS], Google Scholar167https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XptVWltrw%253D&md5=b538dbb714d03d160cf85a59938db843Synthetic Aziridines in Medicinal Chemistry: A Mini-ReviewSingh, Girija S.Mini-Reviews in Medicinal Chemistry (2016), 16 (11), 892-904CODEN: MMCIAE; ISSN:1389-5575. (Bentham Science Publishers Ltd.)Azaheterocyclic compds. are well-known to have diverse types of biol. activity. Among them, azacyclopropanes, commonly referred as aziridines, occupy a prominent place in synthetic org. and medicinal chem. due to its occurrence in natural resources, complexity involved in synthesis due to ring-strain, building blocks in org. synthesis, and its biol. properties. Several novel compds. contg. aziridine ring have been designed and synthesized recently by medicinal chemists for evaluating their biol. profile. A no. of compds. are reported as cysteine protease inhibitors, antibacterial, antifungal, anticancer, antileishmanial, and antimalarial agents. This review article summarizes the biol. activity of such compds. The prepn. of such compds. is also described.
- 168Pitscheider, M.; Mäusbacher, N.; Sieber, S. A. Antibiotic Activity and Target Discovery of Three-Membered Natural Product-Derived Heterocycles in Pathogenic Bacteria. Chem. Sci. 2012, 3, 2035– 2041, DOI: 10.1039/c2sc20290e[Crossref], [CAS], Google Scholar168https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xnt12ru7c%253D&md5=2f89f4792f2ddbd48366cca7beea0904Antibiotic activity and target discovery of three-membered natural product-derived heterocycles in pathogenic bacteriaPitscheider, Maximilian; Maeusbacher, Nina; Sieber, Stephan A.Chemical Science (2012), 3 (6), 2035-2041CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Oxirane, thiirane and aziridine scaffolds represent core motifs in many natural products that usually exhibit their bioactivity by a covalent modification of essential active site residues via a nucleophilic ring opening reaction. Synthesis of these three-membered heterocyclic activity based probes (ABP) was accomplished by robust std. procedures and complemented by a similar set of Michael acceptor systems. Subsequent reactivity studies against the full complement of enzymes in several bacteria revealed an individual fine-tuned affinity and reactivity of each probe that depended on the decoration as well as the heteroatom, resp. Out of many interesting probe-target pairs, we identified thiirane-based compds. that were capable of labeling essential enzymes in S. aureus and L. monocytogenes. Corresponding to the labeling pattern we obsd. an antibiotic effect of these compds. against the clin. relevant pathogens L. monocytogenes as well as multiresistant S. aureus (MRSA).
- 169Lee, M.; Ikejiri, M.; Klimpel, D.; Toth, M.; Espahbodi, M.; Hesek, D.; Forbes, C.; Kumarasiri, M.; Noll, B. C.; Chang, M.; Mobashery, S. Structure–Activity Relationship for Thiirane-Based Gelatinase Inhibitors. ACS Med. Chem. Lett. 2012, 3 (6), 490– 495, DOI: 10.1021/ml300050b[ACS Full Text], [CAS], Google Scholar169https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmsVWgurg%253D&md5=dd16caf1a6f18e07eebfeb53a0a69dd9Structure-Activity Relationship for Thiirane-Based Gelatinase InhibitorsLee, Mijoon; Ikejiri, Masahiro; Klimpel, Dennis; Toth, Marta; Espahbodi, Mana; Hesek, Dusan; Forbes, Christopher; Kumarasiri, Malika; Noll, Bruce C.; Chang, Mayland; Mobashery, ShahriarACS Medicinal Chemistry Letters (2012), 3 (6), 490-495CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)An extensive structure-activity relationship study with the template of 2-(4-phenoxyphenylsulfonylmethyl)thiirane (I), a potent and highly selective inhibitor for human gelatinases, is reported herein. Syntheses of 65 new analogs, each in multistep processes, allowed for exploration of key structural components of the mol. template. This study reveals that the presence of the sulfonylmethylthiirane and the phenoxyphenyl group were important for gelatinase inhibition. However, para- and some meta-substitutions of the terminal Ph ring enhanced inhibitory activity and led to improve metabolic stability. This agrees with the result from metab. studies with compd. I that the primary route of biotransformation is oxidn., mainly at the para position of the Ph ring and the α position of the sulfonyl group in the aliph. side chain.
- 170Harshbarger, W.; Miller, C.; Diedrich, C.; Sacchettini, J. Crystal Structure of the Human 20S Proteasome in Complex with Carfilzomib. Structure 2015, 23 (2), 418– 424, DOI: 10.1016/j.str.2014.11.017[Crossref], [PubMed], [CAS], Google Scholar170https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtF2msbg%253D&md5=0249966770dad6c25b7844fb1cea59b1Crystal Structure of the Human 20S Proteasome in Complex with CarfilzomibHarshbarger, Wayne; Miller, Chase; Diedrich, Chandler; Sacchettini, JamesStructure (Oxford, United Kingdom) (2015), 23 (2), 418-424CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)Proteasome inhibition is highly effective as a treatment for multiple myeloma, and recently carfilzomib was granted US FDA approval for the treatment of relapsed and refractory multiple myeloma. Here, we report the X-ray crystal structure of the human constitutive 20S proteasome with and without carfilzomib bound at 2.9 and 2.6 Å, resp. Our data indicate that the S3 and S4 binding pockets play a pivotal role in carfilzomib's selectivity for chymotrypsin-like sites. Structural comparison with the mouse immunoproteasome crystal structure reveals amino acid substitutions that explain carfilzomib's slight preference for chymotrypsin-like subunits of constitutive proteasomes. In addn., comparison of the human proteasome:carfilzomib complex with the mouse proteasome:PR-957 complex reveals new details that explain why PR-957 is selective for immunoproteasomes. Together, the data presented here support the design of inhibitors for either constitutive or immunoproteasomes, with implications for the treatment of cancers as well as autoimmune and neurodegenerative diseases.
- 171Falagas, M. E.; Vouloumanou, E. K.; Samonis, G.; Vardakas, K. Z. Fosfomycin. Clin. Microbiol. Rev. 2016, 29 (2), 321– 347, DOI: 10.1128/CMR.00068-15[Crossref], [PubMed], [CAS], Google Scholar171https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmsVyhsbY%253D&md5=56a1033535467b306574356bd943fa4bFosfomycinFalagas, Matthew E.; Vouloumanou, Evridiki K.; Samonis, George; Vardakasa, Konstantinos Z.Clinical Microbiology Reviews (2016), 29 (2), 321-347CODEN: CMIREX; ISSN:1098-6618. (American Society for Microbiology)The treatment of bacterial infections suffers from two major problems: spread of multidrug-resistant (MDR) or extensively drug-resistant (XDR) pathogens and lack of development of new antibiotics active against such MDR and XDR bacteria. As a result, physicians have turned to older antibiotics, such as polymyxins, tetracyclines, and aminoglycosides. Lately, due to development of resistance to these agents, fosfomycin has gained attention, as it has remained active against both Gram-pos. and Gram-neg. MDR and XDR bacteria. New data of higher quality have become available, and several issues were clarified further. In this review, we summarize the available fosfomycin data regarding pharmacokinetic and pharmacodynamic properties, the in vitro activity against susceptible and antibiotic-resistant bacteria, mechanisms of resistance and development of resistance during treatment, synergy and antagonism with other antibiotics, clin. effectiveness, and adverse events. Issues that need to be studied further are also discussed.
- 172Kim, D. H.; Lees, W. J.; Kempsell, K. E.; Lane, W. S.; Duncan, K.; Walsh, C. T. Characterization of a Cys115 to Asp Substitution in the Escherichia Coli Cell Wall Biosynthetic Enzyme UDP-GlcNAc Enolpyruvyl Transferase (MurA) That Confers Resistance to Inactivation by the Antibiotic Fosfomycin. Biochemistry 1996, 35 (15), 4923– 4928, DOI: 10.1021/bi952937w[ACS Full Text], [CAS], Google Scholar172https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XhvVCls7w%253D&md5=49606bbdd8809592a5a2c78ece07e4ddCharacterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc Enolpyruvyl Transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycinKim, Dennis H.; Lees, Watson J.; Kempsell, Karen E.; Lane, William S.; Duncan, Kenneth; Walsh, Christopher T.Biochemistry (1996), 35 (15), 4923-8CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The antibiotic fosfomycin inhibits bacterial cell wall biosynthesis by inactivation of UDP-GlcNAc enolpyruvyl transferase (MurA). Prior work has established that Cys115 of Escherichia coli and Enterobacter cloacae MurA is the active site nucleophile alkylated by fosfomycin and implicated this residue in the formation of a covalent phospholactyl-enzyme adduct derived from the substrate, phosphoenolpyruvate (PEP). On the basis of sequencing information from a putative MurA homolog from Mycobacterium tuberculosis, the authors generated a C115D mutant of E. coli MurA that was highly active but fully resistant to time-dependent inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the obsd. reversible competitive inhibition vs. PEP. In contrast to the broad pH-independent behavior of wild-type (WT) MurA, C115D mutant activity titrated across the pH range examd. (pH 5.5-9) with an apparent pKa ∼6, with kcatC115D ranging from ∼10kcatWT at pH 5.5 to <0.1kcatWT at pH 9.0. Km(PEP)C115D was relatively const. in the pH range examd. and increased ∼100-fold relative to Km(PEP)WT. A fosfomycin-resistant C115E mutant with ∼1% activity of the C115D mutant was found to follow a pH dependence similar to that obsd. for C115D MurA. The contrasting pH dependences of WT and C115D MurA were also obsd. in the reaction with the pseudosubstrate, (Z)-3-fluorophosphoenolpyruvate, strongly suggesting a role for Cys/Asp115 as the general acid in the protonation of C-3 of PEP during MurA-catalyzed enol ether transfer. The difference in nucleophilicity between the carboxylate side chains of Asp115 and Glu115 and the thiolate group of Cys115 suggests that covalent enzyme adduct formation is not required for catalytic turnover and, furthermore, provides a chem. rationale for the resistance of the C115D and C115E mutants to fosfomycin inactivation.
- 173Eschenburg, S.; Priestman, M.; Schönbrunn, E. Evidence That the Fosfomycin Target Cys115 in UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Is Essential for Product Release. J. Biol. Chem. 2005, 280 (5), 3757– 3763, DOI: 10.1074/jbc.M411325200[Crossref], [PubMed], [CAS], Google Scholar173https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXovVGmsw%253D%253D&md5=6142eb1dd85ae7d39ef2345c415473b6Evidence that the fosfomycin target Cys115 in UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is essential for product releaseEschenburg, Susanne; Priestman, Melanie; Schoenbrunn, ErnstJournal of Biological Chemistry (2005), 280 (5), 3757-3763CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)MurA (UDP-N-acetylglucosamine enolpyruvyl transferase, EC 2.5.1.7) is an essential enzyme in the biosynthesis of the peptidoglycan layer of the bacterial cell. It provides an attractive template for the design of novel antibiotic drugs and is the target of the naturally occurring antibiotic fosfomycin, which covalently attaches to Cys115 in the active site of the enzyme. Mutations of Cys115 to Asp exist in pathogens such as Mycobacteria or Chlamydia rendering these organisms resistant to fosfomycin. Thus, there is a need for the elucidation of the role of this cysteine in the MurA reaction. We detd. the x-ray structure of the C115S mutant of Enterobacter cloacae MurA, which was crystd. in the presence of the substrates of MurA. The structure depicts the product state of the enzyme with enolpyruvyl-UDP-N-acetylglucosamine (EP-UNAG) and inorg. phosphate (Pi) trapped in the active site. Kinetic anal. revealed that the Cys-to-Ser mutation results in an enzyme that appears to perform a single turnover of the reaction. Opposing the common view of Cys115 as a key residue in the chem. reaction of enolpyruvyl transfer, we now conclude that the wild-type cysteine is essential for product release only. On the basis of a detailed comparison of the product state with the intermediate state and an unliganded state of MurA, we propose that dissocn. of the products is an ordered event with inorg. phosphate leaving first. Phosphate departure appears to trigger a suite of conformational changes, which finally leads to opening of the two-domain structure of MurA and the release of the second product enolpyruvyl-UDP-N-acetylglucosamine.
- 174Porter, N. J.; Christianson, D. W. Binding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8. ACS Chem. Biol. 2017, 12 (9), 2281– 2286, DOI: 10.1021/acschembio.7b00330[ACS Full Text], [CAS], Google Scholar174https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtl2gsrjE&md5=18fbcd74c854bf9183e3d67074338aaeBinding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8Porter, Nicholas J.; Christianson, David W.ACS Chemical Biology (2017), 12 (9), 2281-2286CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Trapoxin A is a microbial cyclic tetrapeptide that is an essentially irreversible inhibitor of class I histone deacetylases (HDACs). The inhibitory warhead is the α,β-epoxyketone side-chain of (2S,9S)-2-amino-8-oxo-9,10-epoxydecanoic acid (L-Aoe), which mimics the side-chain of the HDAC substrate, acetyl-L-lysine. Here, we report the crystal structure of the HDAC8-trapoxin A complex at 1.24 Å resoln., revealing that the ketone moiety of L-Aoe undergoes nucleophilic attack to form a Zn-bound tetrahedral gem-diolate that mimics the tetrahedral intermediate and its flanking transition states in catalysis. Mass spectrometry, activity measurements, and isothermal titrn. calorimetry confirmed that trapoxin A binds tightly (Kd = 3 ± 1 nM) and did not covalently modify the enzyme, so that the epoxide moiety of L-Aoe remained intact. Comparison of the HDAC8-trapoxin A complex with the HDAC6-HC toxin complex provided new insight regarding the inhibitory potency of L-Aoe-contg. natural products against class I and class II HDACs.
- 175Lopus, M.; Smiyun, G.; Miller, H.; Oroudjev, E.; Wilson, L.; Jordan, M. A. Mechanism of Action of Ixabepilone and Its Interactions with the ΒIII-Tubulin Isotype. Cancer Chemother. Pharmacol. 2015, 76 (5), 1013– 1024, DOI: 10.1007/s00280-015-2863-z[Crossref], [PubMed], [CAS], Google Scholar175https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsF2htrvM&md5=0509fbdac680a6e951f27762cc909ef6Mechanism of action of ixabepilone and its interactions with the βIII-tubulin isotypeLopus, Manu; Smiyun, Greg; Miller, Herb; Oroudjev, Emin; Wilson, Leslie; Jordan, Mary AnnCancer Chemotherapy and Pharmacology (2015), 76 (5), 1013-1024CODEN: CCPHDZ; ISSN:0344-5704. (Springer)Ixabepilone (Ixempra, BMS-247550), a semisynthetic analog of epothilone B, is a microtubule-targeted drug in clin. use for treatment of metastatic or locally advanced breast cancer. Ixabepilone's binding and mechanism of action on microtubules and their dynamics, as well as its interactions with isotypically altered microtubules, both in vitro and in tumor cells, have not been described. Microtubules are dynamic polymers of the protein tubulin that function in mitosis, intracellular transport, cell proliferation, and migration. They continually undergo dynamic instability, periods of slow growth and rapid shortening that are crucial to these cell functions. We detd. ixabepilone's microtubule binding and polymn. effects in vitro and also detd. its effects on inhibition of dynamic instability in vitro and in cells, both with and without removal of the βIII isotype of tubulin. The βIII isotype of tubulin is assocd. with drug resistance and tumor aggressivity. We found that removal (in vitro) and knockdown (in cells) of βIII-tubulin led to increased inhibition of microtubule dynamic instability by ixabepilone. Depletion of βIII-tubulin from MCF7 human breast cancer cells also induced increased mitotic arrest by ixabepilone. Thus, βIII-tubulin expression suppresses the antitumor effects of ixabepilone, indicating that increased βIII-tubulin may be an important contributor to the development of resistance to ixabepilone.
- 176Carmi, C.; Cavazzoni, A.; Vezzosi, S.; Bordi, F.; Vacondio, F.; Silva, C.; Rivara, S.; Lodola, A.; Alfieri, R. R.; La Monica, S.; Galetti, M.; Ardizzoni, A.; Petronini, P. G.; Mor, M. Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the Cysteine-Trap Portion. J. Med. Chem. 2010, 53 (5), 2038– 2050, DOI: 10.1021/jm901558p[ACS Full Text], [CAS], Google Scholar176https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhvFOrsbw%253D&md5=5231cae7a553cb1c282e3d09d6901551Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the Cysteine-Trap PortionCarmi, Caterina; Cavazzoni, Andrea; Vezzosi, Stefano; Bordi, Fabrizio; Vacondio, Federica; Silva, Claudia; Rivara, Silvia; Lodola, Alessio; Alfieri, Roberta R.; La Monica, Silvia; Galetti, Maricla; Ardizzoni, Andrea; Petronini, Pier Giorgio; Mor, MarcoJournal of Medicinal Chemistry (2010), 53 (5), 2038-2050CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Irreversible EGFR inhibitors can circumvent acquired resistance to first-generation reversible, ATP-competitive inhibitors in the treatment of non-small-cell lung cancer. They contain both a driver group, which assures target recognition, and a warhead, generally an acrylamide or propargylamide fragment that binds covalently to Cys797 within the kinase domain of EGFR. We performed a systematic exploration of the role for the warhead group, introducing different cysteine-trapping fragments at position 6 of a traditional 4-anilinoquinazoline scaffold. We found that different reactive groups, including epoxyamides (compds. 3-6) and phenoxyacetamides (compds. 7-9), were able to irreversibly inhibit EGFR. In particular, at significant lower concns. than gefitinib (1), (2R,3R)-N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(piperidin-1-ylmethyl)oxirane-2-carboxamide (6) inhibited EGFR autophosphorylation and downstream signaling pathways, suppressed proliferation, and induced apoptosis in gefitinib-resistant NSCLC H1975 cells, harboring the T790M mutation in EGFR.
- 177Klüter, S.; Simard, J. R.; Rode, H. B.; Grütter, C.; Pawar, V.; Raaijmakers, H. C. A.; Barf, T. A.; Rabiller, M.; van Otterlo, W. A. L.; Rauh, D. Characterization of Irreversible Kinase Inhibitors by Directly Detecting Covalent Bond Formation: A Tool for Dissecting Kinase Drug Resistance. ChemBioChem 2010, 11 (18), 2557– 2566, DOI: 10.1002/cbic.201000352[Crossref], [PubMed], [CAS], Google Scholar177https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFahsLrE&md5=5f48cde54e6bad28b4d4f9d47a4e3e3eCharacterization of Irreversible Kinase Inhibitors by Directly Detecting Covalent Bond Formation: A Tool for Dissecting Kinase Drug ResistanceKlueter, Sabine; Simard, Jeffrey R.; Rode, Haridas B.; Gruetter, Christian; Pawar, Vijaykumar; Raaijmakers, Hans C. A.; Barf, Tjeerd A.; Rabiller, Matthias; van Otterlo, Willem A. L.; Rauh, DanielChemBioChem (2010), 11 (18), 2557-2566CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Targeting protein kinases in cancer therapy with irreversible small-mol. inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-assocd. drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biol. and biochem. studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants.
- 178Gehringer, M.; Forster, M.; Laufer, S. A. Solution-Phase Parallel Synthesis of Ruxolitinib-Derived Janus Kinase Inhibitors via Copper-Catalyzed Azide–Alkyne Cycloaddition. ACS Comb. Sci. 2015, 17 (1), 5– 10, DOI: 10.1021/co500122h[ACS Full Text], [CAS], Google Scholar178https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFCisbnI&md5=5d2999fedb2dfef332c2e860a9e376e4Solution-Phase Parallel Synthesis of Ruxolitinib-Derived Janus Kinase Inhibitors via Copper-Catalyzed Azide-Alkyne CycloadditionGehringer, Matthias; Forster, Michael; Laufer, Stefan A.ACS Combinatorial Science (2015), 17 (1), 5-10CODEN: ACSCCC; ISSN:2156-8944. (American Chemical Society)A soln.-phase parallel synthesis of triazole-derived ruxolitinib analogs, e.g. I, was developed in the current study. The method employs copper-catalyzed azide-alkyne cycloaddn. to build up the central triazole template. Product isolation by pptn. and centrifugation is straightforward and yields high purity compds. suited for biol. profiling. A simple protocol for accessing the terminal alkyne precursors in high yields was established and a library of ruxolitinib-like triazoles featuring diverse functional groups was prepd. In addn., a model for the binding mode of ruxolitinib to Janus kinase (JAK) 2 is proposed. In contrast to previous models, the pose explains the compd. selectivity for JAK1/JAK2 and is in accordance with published structure-activity data. On this basis, a structure-based design hypothesis for inverting the selectivity profile of ruxolitinib is deduced. Application of this strategy identified a moderately potent JAK3 inhibitor (35 nM) with high selectivity against other JAKs, potentially exploiting a covalent binding mode.
- 179Forster, M.; Chaikuad, A.; Bauer, S. M.; Holstein, J.; Robers, M. B.; Corona, C. R.; Gehringer, M.; Pfaffenrot, E.; Ghoreschi, K.; Knapp, S.; Laufer, S. A. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket. Cell Chem. Biol. 2016, 23 (11), 1335– 1340, DOI: 10.1016/j.chembiol.2016.10.008[Crossref], [PubMed], [CAS], Google Scholar179https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVeqsLrJ&md5=348c5b5fbfa91fd02fa21d984620ea16Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding PocketForster, Michael; Chaikuad, Apirat; Bauer, Silke M.; Holstein, Julia; Robers, Matthew B.; Corona, Cesear R.; Gehringer, Matthias; Pfaffenrot, Ellen; Ghoreschi, Kamran; Knapp, Stefan; Laufer, Stefan A.Cell Chemical Biology (2016), 23 (11), 1335-1340CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Janus kinases (JAKs) are a family of cytoplasmatic tyrosine kinases that are attractive targets for the development of anti-inflammatory drugs given their roles in cytokine signaling. One question regarding JAKs and their inhibitors that remains under intensive debate is whether JAK inhibitors should be isoform selective. Since JAK3 functions are restricted to immune cells, an isoform-selective inhibitor for JAK3 could be esp. valuable to achieve clin. more useful and precise effects. However, the high degree of structural conservation makes isoform-selective targeting a challenging task. Here, we present picomolar inhibitors with unprecedented kinome-wide selectivity for JAK3. Selectivity was achieved by concurrent covalent reversible targeting of a JAK3-specific cysteine residue and a ligand-induced binding pocket. We confirmed that in vitro activity and selectivity translate well into the cellular environment and suggest that our inhibitors are powerful tools to elucidate JAK3-specific functions.
- 180Rempel, B. P.; Withers, S. G. Covalent Inhibitors of Glycosidases and Their Applications in Biochemistry and Biology. Glycobiology 2008, 18 (8), 570– 586, DOI: 10.1093/glycob/cwn041[Crossref], [PubMed], [CAS], Google Scholar180https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXoslGlsL4%253D&md5=d0a41272cc33a51eaeeb086229ea5789Covalent inhibitors of glycosidases and their applications in biochemistry and biologyRempel, Brian P.; Withers, Stephen G.Glycobiology (2008), 18 (8), 570-586CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)A review. Glycoside hydrolases are important enzymes in a no. of essential biol. processes. Irreversible inhibitors of this class of enzyme have attracted interest as probes of both structure and function. Here, the authors discuss some of the compds. used to covalently modify glycosidases, their use in residue identification, structural and mechanistic investigations, and finally their applications, both in vitro and in vivo, to complex biol. systems.
- 181Povirk, L. F.; Shuker, D. E. DNA Damage and Mutagenesis Induced by Nitrogen Mustards. Mutat. Res., Rev. Genet. Toxicol. 1994, 318 (3), 205– 226, DOI: 10.1016/0165-1110(94)90015-9[Crossref], [PubMed], [CAS], Google Scholar181https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXitlaktbo%253D&md5=e29f751b42550ae503639e6b2fc0fdd1DNA damage and mutagenesis induced by nitrogen mustardsPovirk, Lawrence F.; Shuker, David E.Mutation Research, Reviews in Genetic Toxicology (1994), 318 (3), 205-26CODEN: MRRTEP; ISSN:0165-1110. (Elsevier B.V.)A review with many refs. The nitrogen mustards are bifunctional alkylating agents which, although used extensively in cancer chemotherapy, are themselves highly carcinogenic. All nitrogen mustards induce monofunctional guanine-N7 adducts, as well as interstrand N7-N7 crosslinks involving the two guanines in GNC·GNC (5'→3'/5'→3') sequences. In addn., the arom. mustards melphalan and chlorambucil also induce substantial alkylation at adenine N3, while cyclophosphamide forms phosphotriesters with relatively high frequency. Nitrogen mustards are genotoxic in virtually every assay, and produce a wide array of mutations, including base substitutions at both G·C and A·T base pairs, intragenic as well as multilocus deletions, and chromosomal rearrangements. Mutational spectra generated by these agents in various model systems vary widely, and no single lesion has been implicated as being primarily responsible for mustard-induced mutagenesis. On the contrary, adducts of both adenine and guanine, and monofunctional as well as bifunctional adducts, appear to be involved. Further, it is still not known which types of mutation are responsible for mustard-induced cancers, since no genes have yet been identified which are consistently altered in these malignancies.
- 182McGregor, L. M.; Jenkins, M. L.; Kerwin, C.; Burke, J. E.; Shokat, K. M. Expanding the Scope of Electrophiles Capable of Targeting K-Ras Oncogenes. Biochemistry 2017, 56 (25), 3178– 3183, DOI: 10.1021/acs.biochem.7b00271[ACS Full Text], [CAS], Google Scholar182https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVGrsbnI&md5=0b25b65210248a4cad2916efba90ddaeExpanding the Scope of Electrophiles Capable of Targeting K-Ras OncogenesMcGregor, Lynn M.; Jenkins, Meredith L.; Kerwin, Caitlin; Burke, John E.; Shokat, Kevan M.Biochemistry (2017), 56 (25), 3178-3183CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)There is growing interest in reversible and irreversible covalent inhibitors that target noncatalytic amino acids in target proteins. With a goal of targeting oncogenic K-Ras variants (e.g., G12D) by expanding the types of amino acids that can be targeted by covalent inhibitors, we survey a set of electrophiles for their ability to label carboxylates. We functionalized an optimized ligand for the K-Ras switch II pocket with a set of electrophiles previously reported to react with carboxylates and characterized the ability of these compds. to react with model nucleophiles and oncogenic K-Ras proteins. Here, we report that aziridines and stabilized diazo groups preferentially react with free carboxylates over thiols. Although we did not identify a warhead that potently labels K-Ras G12D, we were able to study the interactions of many electrophiles with K-Ras, as most of the electrophiles rapidly label K-Ras G12C. We characterized the resulting complexes by crystallog., hydrogen/deuterium exchange, and differential scanning fluorometry. Our results both demonstrate the ability of a noncatalytic cysteine to react with a diverse set of electrophiles and emphasize the importance of proper spatial arrangements between a covalent inhibitor and its intended nucleophile. We hope that these results can expand the range of electrophiles and nucleophiles of use in covalent protein modulation.
- 183Ray, S.; Kreitler, D. F.; Gulick, A. M.; Murkin, A. S. The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition. ACS Chem. Biol. 2018, 13 (6), 1470– 1473, DOI: 10.1021/acschembio.8b00225[ACS Full Text], [CAS], Google Scholar183https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpsl2nsLc%253D&md5=d2dc8f255fd7e5f7537c57a0c5a3343eThe nitro group as a masked electrophile in covalent enzyme inhibitionRay, Sneha; Kreitler, Dale F.; Gulick, Andrew M.; Murkin, Andrew S.ACS Chemical Biology (2018), 13 (6), 1470-1473CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)We report the unprecedented reaction between a nitroalkane and an active-site cysteine residue to yield a thiohydroximate adduct. Structural and kinetic evidence suggests the nitro group is activated by conversion to its nitronic acid tautomer within the active site. The nitro group, therefore, shows promise as a masked electrophile in the design of covalent inhibitors targeting binding pockets with appropriately placed cysteine and general acid residues.
- 184Moynihan, M. M.; Murkin, A. S. Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate. Biochemistry 2014, 53 (1), 178– 187, DOI: 10.1021/bi401432t[ACS Full Text], [CAS], Google Scholar184https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFOiurvM&md5=07384a8e75df3a87db4ed9e0010b12bcCysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-NitropropionateMoynihan, Margaret M.; Murkin, Andrew S.Biochemistry (2014), 53 (1), 178-187CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Isocitrate lyase (ICL) catalyzes the reversible cleavage of isocitrate into succinate and glyoxylate. It is the first committed step in the glyoxylate cycle used by some organisms, including Mycobacterium tuberculosis, where it has been shown to be essential for cell survival during chronic infection. The pH-rate and pD-rate profiles measured in the direction of isocitrate synthesis revealed solvent kinetic isotope effects (KIEs) of 1.7 ± 0.4 for D2OV and 0.56 ± 0.07 for D2O(V/Ksuccinate). Whereas the D2OV is consistent with partially rate-limiting proton transfer during formation of the hydroxyl group of isocitrate, the large inverse D2O(V/Ksuccinate) indicates that substantially different kinetic parameters exist when the enzyme is satd. with succinate. Inhibition by 3-nitropropionate (3-NP), a succinate analog, was found to proceed through an unusual double slow-onset process featuring formation of a complex with a Ki of 3.3 ± 0.2 μM during the first minute, followed by formation of a final complex with a Ki* of 44 ± 10 nM over the course of several minutes to hours. Stopped-flow measurements during the first minute revealed an apparent solvent KIE of 0.40 ± 0.03 for assocn. and unity for dissocn. In contrast, itaconate, a succinate analog lacking an acidic α-proton, did not display slow-binding behavior and yielded a D2OKi of 1.0 ± 0.2. These results support a common mechanism for catalysis with succinate and inhibition by 3-NP featuring (1) an unfavorable prebinding isomerization of the active site Cys191-His193 pair to the thiolate-imidazolium form, a process that is favored in D2O, and (2) the transfer of a proton from succinate or 3-NP to Cys191. These findings also indicate that propionate-3-nitronate, which is the conjugate base of 3-NP and the "true inhibitor" of ICL, does not bind directly and must be generated enzymically.
- 185Krenske, E. H.; Petter, R. C.; Houk, K. N. Kinetics and Thermodynamics of Reversible Thiol Additions to Mono- and Diactivated Michael Acceptors: Implications for the Design of Drugs That Bind Covalently to Cysteines. J. Org. Chem. 2016, 81 (23), 11726– 11733, DOI: 10.1021/acs.joc.6b02188[ACS Full Text], [CAS], Google Scholar185https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhsl2gtbbK&md5=64cf60c3ab5b5778b28cbd0aed528d37Kinetics and Thermodynamics of Reversible Thiol Additions to Mono- and Diactivated Michael Acceptors: Implications for the Design of Drugs That Bind Covalently to CysteinesKrenske, Elizabeth H.; Petter, Russell C.; Houk, K. N.Journal of Organic Chemistry (2016), 81 (23), 11726-11733CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)Addns. of cysteine thiols to Michael acceptors underpin the mechanism of action of several covalent drugs (e.g., afatinib, osimertinib, ibrutinib, neratinib, and CC-292). Reversible Michael acceptors were reported in which an addnl. electron-withdrawing group was added at the α-carbon of a Michael acceptor. The authors have performed d. functional theory calcns. to det. why thiol addns. to these Michael acceptors are reversible. The α-EWG group stabilizes the anionic transition state and intermediate of the Michael addn., but less intuitively, it destabilizes the neutral adduct. This makes the reverse reaction (elimination) both faster and more thermodynamically favorable. For thiol addn. to be reversible, the Michael acceptor must also contain a suitable substituent on the β-carbon, such as an aryl or branched alkyl group. Computations explain how these structural elements contribute to reversibility and the ability to tune the binding affinities and the residence times of covalent inhibitors.
- 186Krishnan, S.; Miller, R. M.; Tian, B.; Mullins, R. D.; Jacobson, M. P.; Taunton, J. Design of Reversible, Cysteine-Targeted Michael Acceptors Guided by Kinetic and Computational Analysis. J. Am. Chem. Soc. 2014, 136 (36), 12624– 12630, DOI: 10.1021/ja505194w[ACS Full Text], [CAS], Google Scholar186https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVWrtbvK&md5=de052bac07c8920cb807c05f71f4f95dDesign of Reversible, Cysteine-Targeted Michael Acceptors Guided by Kinetic and Computational AnalysisKrishnan, Shyam; Miller, Rand M.; Tian, Boxue; Mullins, R. Dyche; Jacobson, Matthew P.; Taunton, JackJournal of the American Chemical Society (2014), 136 (36), 12624-12630CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacol. potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo β-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our exptl. and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.
- 187Smith, S.; Keul, M.; Engel, J.; Basu, D.; Eppmann, S.; Rauh, D. Characterization of Covalent-Reversible EGFR Inhibitors. ACS Omega 2017, 2 (4), 1563– 1575, DOI: 10.1021/acsomega.7b00157[ACS Full Text], [CAS], Google Scholar187https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmt1KqsLg%253D&md5=695eb8fbc519ba2fba3ff7d5296b4116Characterization of Covalent-Reversible EGFR InhibitorsSmith, Steven; Keul, Marina; Engel, Julian; Basu, Debjit; Eppmann, Simone; Rauh, DanielACS Omega (2017), 2 (4), 1563-1575CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)Within the spectrum of kinase inhibitors, covalent-reversible inhibitors (CRIs) provide a valuable alternative approach to classical covalent inhibitors. This special class of inhibitors can be optimized for an extended drug-target residence time. For CRIs, it was shown that the fast addn. of thiols to electron-deficient olefins leads to a covalent bond that can break reversibly under proteolytic conditions. Research groups are just beginning to include CRIs in their arsenal of compd. classes, and, with that, the understanding of this interesting set of chem. warheads is growing. However, systems to assess both characteristics of the covalent-reversible bond in a simple exptl. setting are sparse. Here, we have developed an efficient methodol. to characterize the covalent and reversible properties of CRIs and to investigate their potential in targeting clin. relevant variants of the receptor tyrosine kinase EGFR.
- 188Forster, M.; Chaikuad, A.; Dimitrov, T.; Döring, E.; Holstein, J.; Berger, B.-T.; Gehringer, M.; Ghoreschi, K.; Müller, S.; Knapp, S.; Laufer, S. A. Development, Optimization, and Structure–Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4-d]Pyrrolo[2,3-b]Pyridine Scaffold. J. Med. Chem. 2018, 61 (12), 5350– 5366, DOI: 10.1021/acs.jmedchem.8b00571[ACS Full Text], [CAS], Google Scholar188https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVGjsrbN&md5=8acdec137df746f854bbee75a10d5f70Development, Optimization, and Structure-Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4-d]pyrrolo[2,3-b]pyridine ScaffoldForster, Michael; Chaikuad, Apirat; Dimitrov, Teodor; Doering, Eva; Holstein, Julia; Berger, Benedict-Tilman; Gehringer, Matthias; Ghoreschi, Kamran; Mueller, Susanne; Knapp, Stefan; Laufer, Stefan A.Journal of Medicinal Chemistry (2018), 61 (12), 5350-5366CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Janus kinases are major drivers of immune signaling and have been the focus of anti-inflammatory drug discovery for more than a decade. Because of the invariable colocalization of JAK1 and JAK3 at cytokine receptors, the question if selective JAK3 inhibition is sufficient to effectively block downstream signaling has been highly controversial. Recently, we discovered the covalent-reversible JAK3 inhibitor FM-381 (23) featuring high isoform and kinome selectivity. Crystallog. revealed that this inhibitor induces an unprecedented binding pocket by interactions of a nitrile substituent with arginine residues in JAK3. Herein, we describe detailed structure-activity relationships necessary for induction of the arginine pocket and the impact of this structural change on potency, isoform selectivity, and efficacy in cellular models. Furthermore, we evaluated the stability of this novel inhibitor class in in vitro metabolic assays and were able to demonstrate an adequate stability of key compd. 23 for in vivo use.
- 189Cheung, S. T.; Miller, M. S.; Pacoma, R.; Roland, J.; Liu, J.; Schumacher, A. M.; Hsieh-Wilson, L. C. Discovery of a Small-Molecule Modulator of Glycosaminoglycan Sulfation. ACS Chem. Biol. 2017, 12 (12), 3126– 3133, DOI: 10.1021/acschembio.7b00885[ACS Full Text], [CAS], Google Scholar189https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslGrsbfE&md5=4626679495c584d0ce81daa64a920b7aDiscovery of a Small-Molecule Modulator of Glycosaminoglycan SulfationCheung, Sheldon T.; Miller, Michelle S.; Pacoma, Reynand; Roland, Jason; Liu, Jian; Schumacher, Andrew M.; Hsieh-Wilson, Linda C.ACS Chemical Biology (2017), 12 (12), 3126-3133CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Glycosaminoglycans (GAGs) play crit. roles in diverse processes ranging from viral infection to neuroregeneration. Their regiospecific sulfation patterns, which are generated by sulfotransferases, are key structural determinants that underlie their biol. activity. Small-mol. modulators of these sulfotransferases could serve as powerful tools for understanding the physiol. functions of GAGs, as well as potential therapeutic leads for human diseases. Here, the authors report the development of the first cell-permeable, small-mol. inhibitor selective for GAG sulfotransferases, which was obtained using a high-throughput screen targeted against Chst15, the sulfotransferase responsible for biosynthesis of chondroitin sulfate-E (CS-E). The authors demonstrate that the mol. specifically inhibits GAG sulfotransferases in vitro, decreases CS-E and overall sulfation levels on cell-surface and secreted chondroitin sulfate proteoglycans (CSPGs), and reverses CSPG-mediated inhibition of axonal growth. These studies pave the way toward a new set of pharmacol. tools for interrogating GAG sulfation-dependent processes and may represent a novel therapeutic approach for neuroregeneration.
- 190Cully, M. Rational Drug Design: Tuning Kinase Inhibitor Residence Time. Nat. Rev. Drug Discovery 2015, 14, 457, DOI: 10.1038/nrd4673[Crossref], [PubMed], [CAS], Google Scholar190https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFeju7nI&md5=3bcab237493059803552dafd0e4fc6e5Rational drug design Tuning kinase inhibitor residence timeCully, MeganNature Reviews Drug Discovery (2015), 14 (7), 457CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)There is no expanded citation for this reference.
- 191Langrish, C. L.; Bradshaw, J. M.; Owens, T. D.; Campbell, R. L.; Francesco, M. R.; Karr, D. E.; Murray, S. K.; Quesenberry, R. C.; Smith, P. F.; Taylor, M. D.; Zhu, J.; Nunn, P. A.; Gourlay, S. G. PRN1008, a Reversible Covalent BTK Inhibitor in Clinical Development for Immune Thrombocytopenic Purpura. Blood 2017, 130, 1052
- 192Masjedizadeh, M. R.; Gourlay, S. Salts and Solid Form of a Btk Inhibitor. WO2015127310, August 28, 2015.
- 193Holm, K. J.; Spencer, C. M. Entacapone. Drugs 1999, 58 (1), 159– 177, DOI: 10.2165/00003495-199958010-00017[Crossref], [PubMed], [CAS], Google Scholar193https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXltFOju7Y%253D&md5=3285c98f7cdf43918bad3fded4a39bc5Entacapone: a review of its use in Parkinson's diseaseHolm, Kristin J.; Spencer, Caroline M.Drugs (1999), 58 (1), 159-177CODEN: DRUGAY; ISSN:0012-6667. (Adis International Ltd.)A review with 58 refs. Entacapone is a potent and specific peripheral catechol-O-methyltransferase (COMT) inhibitor. It has been shown to improve the clin. benefits of levodopa plus an arom. L-amino acid decarboxylase inhibitor (AADC) when given to patients with Parkinson's disease and end-of-dose deterioration in the response to levodopa (the "wearing off" phenomenon). The efficacy of entacapone is currently being assessed in patients with stable Parkinson's disease. In 2 well conducted trials of 6 mo' duration and smaller short term studies, treatment with entacapone (200mg with each dose of levodopa/AADC inhibitor) was assocd. with significant increases in daily "on" time and decreases in "off" time. Changes in Unified Parkinson's Disease Rating Scale (UPDRS) scores concurred with changes in "on" and "off" times: entacapone improved total, activities of daily living and motor function scores, but it had no effect on mentation scores. Entacapone also provided benefits when given with controlled release levodopa/AADC inhibitor or with std. levodopa/AADC inhibitor and selegiline in small trials. Dopaminergic events, including dyskinesia and nausea, are among the most common events with entacapone, and are related to the drug's ability to potentiate the effects of levodopa. Diarrhea, abdominal pain, constipation and urine discoloration are the most common nondopaminergic events, although the latter event is the only one to occur consistently more frequently with entacapone than with placebo. However, adverse events of any type infrequently led to study discontinuation. The efficacy and tolerability of entacapone administered with levodopa/AADC inhibitor have not yet been compared with those of other strategies for the treatment of Parkinson's disease. However, once the decision to initiate levodopa therapy has been made, studies generally support the use of entacapone as an adjunct to levodopa in patients with Parkinson's disease and the "wearing off" phenomenon.
- 194Mendgen, T.; Steuer, C.; Klein, C. D. Privileged Scaffolds or Promiscuous Binders: A Comparative Study on Rhodanines and Related Heterocycles in Medicinal Chemistry. J. Med. Chem. 2012, 55 (2), 743– 753, DOI: 10.1021/jm201243p[ACS Full Text], [CAS], Google Scholar194https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVGkt7nN&md5=6a94d097f44bdbaaec9a56fd6b371521Privileged Scaffolds or Promiscuous Binders: A Comparative Study on Rhodanines and Related Heterocycles in Medicinal ChemistryMendgen, Thomas; Steuer, Christian; Klein, Christian D.Journal of Medicinal Chemistry (2012), 55 (2), 743-753CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Rhodanines and related five-membered heterocycles with multiple heteroatoms have recently gained a reputation of being unselective compds. that appear as "frequent hitters" in screening campaigns and therefore have little value in drug discovery. However, this judgment appears to be based mostly on anecdotal evidence. Having identified various rhodanines and related compds. in screening campaigns, we decided to perform a systematic study on their promiscuity. An amt. of 163 rhodanines, hydantoins, thiohydantoins, and thiazolidinediones were synthesized and tested against several targets. The compds. were also characterized with respect to aggregation and electrophilic reactivity, and the binding modes of rhodanines and related compds. in published x-ray cocrystal structures were analyzed. The results indicate that the exocyclic, double bonded sulfur atom in rhodanines and thiohydantoins, in addn. to other structural features, offers a particularly high d. of interaction sites for polar interactions and hydrogen bonds. This causes a promiscuous behavior at concns. in the "screening range" but should not be regarded as a general knockout criterion that excludes such screening hits from further development. It is suggested that special criteria for target affinity and selectivity are applied to these classes of compds. and that their exceptional and potentially valuable biomol. binding properties are consequently exploited in a useful way.
- 195Schneider, T. H.; Rieger, M.; Ansorg, K.; Sobolev, A. N.; Schirmeister, T.; Engels, B.; Grabowsky, S. Vinyl Sulfone Building Blocks in Covalently Reversible Reactions with Thiols. New J. Chem. 2015, 39 (7), 5841– 5853, DOI: 10.1039/C5NJ00368G[Crossref], [CAS], Google Scholar195https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVWjt7jE&md5=2f65514bf92ec07ba1d344aa9b8a7599Vinyl sulfone building blocks in covalently reversible reactions with thiolsSchneider, Thomas H.; Rieger, Max; Ansorg, Kay; Sobolev, Alexandre N.; Schirmeister, Tanja; Engels, Bernd; Grabowsky, SimonNew Journal of Chemistry (2015), 39 (7), 5841-5853CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)In the present study we use quantum-chem. calcns. to investigate how the reactivity of vinyl sulfone-based compds. can be modified from an irreversible to a reversible reaction with thiols. Based on the predictions from theory, an array of nine different vinyl sulfones with systematically varying substitution pattern was synthesized and their crystal structures were detd. Subsequent Hirshfeld surface analyses employing the principle of electrostatic complementarity aid the understanding of the crystal packing of the synthesized compds. Reactivity studies against the nucleophile 2-phenylethanethiol mirror the properties predicted by the quantum-chem. computations in soln.
- 196Siklos, M.; BenAissa, M.; Thatcher, G. R. J. Cysteine Proteases as Therapeutic Targets: Does Selectivity Matter? A Systematic Review of Calpain and Cathepsin Inhibitors. Acta Pharm. Sin. B 2015, 5 (6), 506– 519, DOI: 10.1016/j.apsb.2015.08.001[Crossref], [PubMed], [CAS], Google Scholar196https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28rlslWjsg%253D%253D&md5=cbd246a39984c0d9e3a32fd26db8902dCysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitorsSiklos Marton; BenAissa Manel; Thatcher Gregory R JActa pharmaceutica Sinica. B (2015), 5 (6), 506-19 ISSN:2211-3835.Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.
- 197Cleary, J. A.; Doherty, W.; Evans, P.; Malthouse, J. P. G. Quantifying Tetrahedral Adduct Formation and Stabilization in the Cysteine and the Serine Proteases. Biochim. Biophys. Acta, Proteins Proteomics 2015, 1854, 1382– 1391, DOI: 10.1016/j.bbapap.2015.07.006[Crossref], [PubMed], [CAS], Google Scholar197https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFOktL%252FN&md5=f590715d70dc21d5553ba2c148a46796Quantifying tetrahedral adduct formation and stabilization in the cysteine and the serine proteasesCleary, Jennifer A.; Doherty, William; Evans, Paul; Malthouse, J. Paul G.Biochimica et Biophysica Acta, Proteins and Proteomics (2015), 1854 (10_Part_A), 1382-1391CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)Two new papain inhibitors have been synthesized where the terminal α-carboxyl groups of Z-Phe-Ala-COOH and Ac-Phe-Gly-COOH have been replaced by a proton to give Z-Phe-Ala-H and Ac-Phe-Gly-H. The authors show that for papain, replacing the terminal carboxylate group of a peptide inhibitor with a H atom decreased binding 3- to 4-fold, while replacing an aldehyde or glyoxal group with a H atom decreased binding by 300,000- to 1,000,000-fold. Thiohemiacetal formation by papain with aldehyde or glyoxal inhibitors was shown to be ∼10,000-fold more effective than hemiacetal or hemiketal formation with chymotrypsin. It was shown using effective molarities, that for papain, thiohemiacetal stabilization was more effective with aldehyde inhibitors than with glyoxal inhibitors. The effective molarity obtained when papain was inhibited by an aldehyde inhibitor was similar to the effective molarity obtained when chymotrypsin was inhibited by glyoxal inhibitors showing that both enzymes can stabilize tetrahedral adducts by similar amts. Therefore, the greater potency of aldehyde and glyoxal inhibitors with papain was not due to greater thiohemiacetal stabilization by papain compared to the hemiketal and hemiacetal stabilization by chymotrypsin, instead it reflected the greater intrinsic reactivity of the catalytic SH group of papain compared to the catalytic OH group of chymotrypsin. It was argued that while the hemiacetals and thiohemiacetals formed with the serine and cysteine proteases, resp., can mimic the catalytic tetrahedral intermediate they are also analogs of the productive and non-productive acyl intermediates that can be formed with the cysteine and serine proteases.
- 198Sanches, M.; Duffy, N. M.; Talukdar, M.; Thevakumaran, N.; Chiovitti, D.; Canny, M. D.; Lee, K.; Kurinov, I.; Uehling, D.; Al-awar, R.; Poda, G.; Prakesch, M.; Wilson, B.; Tam, V.; Schweitzer, C.; Toro, A.; Lucas, J. L.; Vuga, D.; Lehmann, L.; Durocher, D.; Zeng, Q.; Patterson, J. B.; Sicheri, F. Structure and Mechanism of Action of the Hydroxy–Aryl–Aldehyde Class of IRE1 Endoribonuclease Inhibitors. Nat. Commun. 2014, 5, 4202, DOI: 10.1038/ncomms5202[Crossref], [PubMed], [CAS], Google Scholar198https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksVertrk%253D&md5=bbdaaf0fad0ae104e3b1790bcc50df2bStructure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitorsSanches, Mario; Duffy, Nicole M.; Talukdar, Manisha; Thevakumaran, Nero; Chiovitti, David; Canny, Marella D.; Lee, Kenneth; Kurinov, Igor; Uehling, David; Al-Awar, Rima; Poda, Gennadiy; Prakesch, Michael; Wilson, Brian; Tam, Victor; Schweitzer, Colleen; Toro, Andras; Lucas, Julie L.; Vuga, Danka; Lehmann, Lynn; Durocher, Daniel; Zeng, Qingping; Patterson, John B.; Sicheri, FrankNature Communications (2014), 5 (), 4202CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Arom. ring systems contg. hydroxy-aldehyde moieties, termed hydroxy-aryl-aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chem. series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase-active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a hydrogen bond with Tyr892. Structure-activity studies and mutational anal. of contact residues define the optimal chem. space of inhibitors and validate the inhibitor-binding site. These studies lay the foundation for understanding both the biochem. and cellular functions of IRE1α using small mol. inhibitors and suggest new avenues for inhibitor design.
- 199Larraufie, M.-H.; Yang, W. S.; Jiang, E.; Thomas, A. G.; Slusher, B. S.; Stockwell, B. R. Incorporation of Metabolically Stable Ketones into a Small Molecule Probe to Increase Potency and Water Solubility. Bioorg. Med. Chem. Lett. 2015, 25 (21), 4787– 4792, DOI: 10.1016/j.bmcl.2015.07.018[Crossref], [PubMed], [CAS], Google Scholar199https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFOktL%252FF&md5=1816055cbe6493ea7336df8b59c18a35Incorporation of metabolically stable ketones into a small molecule probe to increase potency and water solubilityLarraufie, Marie-Helene; Yang, Wan Seok; Jiang, Elise; Thomas, Ajit G.; Slusher, Barbara S.; Stockwell, Brent R.Bioorganic & Medicinal Chemistry Letters (2015), 25 (21), 4787-4792CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Introducing a reactive carbonyl to a scaffold that does not otherwise have an electrophilic functionality to create a reversible covalent inhibitor is a potentially useful strategy for enhancing compd. potency. However, aldehydes are metabolically unstable, which precludes the use of this strategy for compds. to be tested in animal models or in human clin. studies. To overcome this limitation, the authors designed ketone-based functionalities capable of forming reversible covalent adducts, while displaying high metabolic stability, and imparting improved water soly. to their pendant scaffold. The authors tested this strategy on the ferroptosis inducer and exptl. therapeutic erastin, and obsd. substantial increases in compd. potency. In particular, a new carbonyl erastin analog, termed IKE, displayed improved potency, soly. and metabolic stability, thus representing an ideal candidate for future in vivo cancer therapeutic applications.
- 200Cross, B. C. S.; Bond, P. J.; Sadowski, P. G.; Jha, B. K.; Zak, J.; Goodman, J. M.; Silverman, R. H.; Neubert, T. A.; Baxendale, I. R.; Ron, D.; Harding, H. P. The Molecular Basis for Selective Inhibition of Unconventional MRNA Splicing by an IRE1-Binding Small Molecule. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (15), E869– E878, DOI: 10.1073/pnas.1115623109[Crossref], [PubMed], [CAS], Google Scholar200https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmtVCgtb8%253D&md5=f6a05686d1b4a8a25d33fea41270f3eeThe molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small moleculeCross, Benedict C. S.; Bond, Peter J.; Sadowski, Pawel G.; Jha, Babal Kant; Zak, Jaroslav; Goodman, Jonathan M.; Silverman, Robert H.; Neubert, Thomas A.; Baxendale, Ian R.; Ron, David; Harding, Heather P.Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (15), E869-E878, SE869/1-SE869/13CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degrdn. of diverse membrane-bound mRNAs. We report on the identification of a small mol. inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degrdn. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chem. reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathol. settings.
- 201Knoepfel, T.; Furet, P.; Mah, R.; Buschmann, N.; Leblanc, C.; Ripoche, S.; Graus-Porta, D.; Wartmann, M.; Galuba, I.; Fairhurst, R. A. 2-Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4. ACS Med. Chem. Lett. 2018, 9 (3), 215– 220, DOI: 10.1021/acsmedchemlett.7b00485[ACS Full Text], [CAS], Google Scholar201https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit1Srsb4%253D&md5=fd43811b8a9e5f7a02f27096569423f82-Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4Knoepfel, Thomas; Furet, Pascal; Mah, Robert; Buschmann, Nicole; Leblanc, Catherine; Ripoche, Sebastien; Graus-Porta, Diana; Wartmann, Markus; Galuba, Inga; Fairhurst, Robin A.ACS Medicinal Chemistry Letters (2018), 9 (3), 215-220CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)As part of a project to identify FGFR4 selective inhibitors, scaffold morphing of a 2-formylquinoline amide hit identified series of 2-formylpyridine ureas (2-FPUs) with improved potency and physicochem. properties. In particular, tetrahydronaphthyridine urea analogs with cellular activities below 30 nM have been identified. Consistent with the hypothesized reversible-covalent mechanism of inhibition, the 2-FPUs exhibited slow binding kinetics, and the aldehyde, as the putative electrophile, could be demonstrated to be a key structural element for activity.
- 202LoPachin, R. M.; Gavin, T. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective. Chem. Res. Toxicol. 2014, 27 (7), 1081– 1091, DOI: 10.1021/tx5001046[ACS Full Text], [CAS], Google Scholar202https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpsVensrg%253D&md5=e72707796ea59ccaff0283848ebbdf76Molecular Mechanisms of Aldehyde Toxicity: A Chemical PerspectiveLoPachin, Richard M.; Gavin, TerrenceChemical Research in Toxicology (2014), 27 (7), 1081-1091CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)A review. Aldehydes are electrophilic compds. to which humans are pervasively exposed. Despite a significant health risk due to exposure, the mechanisms of aldehyde toxicity are poorly understood. This ambiguity is likely due to the structural diversity of aldehyde derivs. and corresponding differences in chem. reactions and biol. targets. To gain mechanistic insight, the authors have used parameters based on the hard and soft, acids and bases (HSAB) theory to profile the different aldehyde subclasses with respect to electronic character (softness, hardness), electrophilic reactivity (electrophilic index), and biol. nucleophilic targets. The authors' analyses indicate that short chain aldehydes and longer chain satd. alkanals are hard electrophiles that cause toxicity by forming adducts with hard biol. nucleophiles, e.g., primary nitrogen groups on lysine residues. In contrast, α,β-unsatd. carbonyl derivs., alkenals, and the α-oxoaldehydes are soft electrophiles that preferentially react with soft nucleophilic thiolate groups on cysteine residues. The aldehydes can therefore be grouped into subclasses according to common electronic characteristics (softness/hardness) and mol. mechanisms of toxicity. As the authors will discuss, the toxic potencies of these subgroups are generally related to corresponding electrophilicities. For some aldehydes, however, predictions of toxicity based on electrophilicity are less accurate due to inherent physicochem. variables that limit target accessibility, e.g., steric hindrance and soly. The unsatd. aldehydes are also members of the conjugated type-2 alkene chem. class that includes α,β-unsatd. amide, ketone, and ester derivs. Type-2 alkenes are electrophiles of varying softness and electrophilicity that share a common mechanism of toxicity. Therefore, exposure to an environmental mixt. of unsatd. carbonyl derivs. could cause "type-2 alkene toxicity" through additive interactions. Finally, the authors propose that environmentally derived aldehydes can accelerate diseases by interacting with endogenous aldehydes generated during oxidative stress. This review provides a basis for understanding aldehyde mechanisms and environmental toxicity through the context of electronic structure, electrophilicity, and nucleophile target selectivity.
- 203Caraballo, R.; Dong, H.; Ribeiro, J. P.; Jiménez-Barbero, J.; Ramström, O. Direct STD NMR Identification of β-Galactosidase Inhibitors from a Virtual Dynamic Hemithioacetal System. Angew. Chem., Int. Ed. 2010, 49 (3), 589– 593, DOI: 10.1002/anie.200903920[Crossref], [CAS], Google Scholar203https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktlaksw%253D%253D&md5=012f6210d6e743fe5dd958cc4500d937Direct STD NMR Identification of β-Galactosidase Inhibitors from a Virtual Dynamic Hemithioacetal SystemCaraballo, Remi; Dong, Hai; Ribeiro, Joao P.; Jimenez-Barbero, Jesus; Ramstroem, OlofAngewandte Chemie, International Edition (2010), 49 (3), 589-593, S589/1-S589/20CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Herein we show that hemithioacetal (HTA) formation is a fast and efficient reversible reaction that can be used to generate systems of potential enzyme inhibitors in aq. media under neutral conditions. These systems were also subjected to direct binding anal. by satn. transfer difference (STD) NMR spectroscopy, a rapid and efficient technique for protein-ligand binding studies, in which the optimal HTA interactions with the biol. target can be readily deduced. We have successfully demonstrated, for the first time, hemithioacetal formation applied to dynamic combinatorial system generation in aq. media. Equil. formation using this chem. proved very rapid and resulted in truly virtual dynamic systems in which 1H NMR analyses confirmed the presence of transient HTA constituents. It has further been demonstrated that 1H STD NMR spectroscopy can be used for efficient and direct in situ identification of the best enzyme binders of the virtual dynamic system. Inhibition studies unequivocally support the 1H STD NMR.exptl. data and enable the distinction of different β-galactosidase inhibitors and nonspecific binders.
- 204Buschmann, N.; Fairhurst, R. A.; Knoepfel, T.; Furet, P.; Leblanc, C.; Mah, R.; Kiffe, M.; Graus-Porta, D.; Weiss, A.; Kinyamu-Akunda, J.; Wartmann, M.; Trappe, J.; Gabriel, T. R.; Hofmann, F.; Sellers, W. R. A Reversible Covalent Approach to Selective FGFR4 Inhibition; Book of Abstracts—Frontiers in Medicinal Chemistry Symposium, Jena, 2018; p 26.
- 205Otto, H.-H.; Schirmeister, T. Cysteine Proteases and Their Inhibitors. Chem. Rev. 1997, 97 (1), 133– 172, DOI: 10.1021/cr950025u[ACS Full Text], [CAS], Google Scholar205https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXktFOhsA%253D%253D&md5=0c38c0f09f14ad664b6ca707ce08a5b9Cysteine Proteases and Their InhibitorsOtto, Hans-Hartwig; Schirmeister, TanjaChemical Reviews (Washington, D. C.) (1997), 97 (1), 133-171CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with 599 refs. Some typical examples of cysteine proteases from different sources are characterized in terms of property, structure and specificity. Possible functions of human and mammalian cysteine proteases, lysosomal cathepsins and cytoplasmic calpains are discussed, as well as their role in pathol. processes. In addn. to the endogenous inhibitors, the cystatins and calpastatins, the currently available synthetic inhibitors are also described together with their structures, reaction mechanisms, activities and selectivities. Potential medical applications of cysteine protease inhibitors are discussed. Particular emphasis is given to comparison of cysteine proteases with serine proteases, due to their similar proteolytic mechanisms.
- 206Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.; Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.; Huang, Q.; Han, S.-P.; Abboa-Offei, B.; Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.; Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. Discovery and Preclinical Profile of Saxagliptin (BMS-477118): A Highly Potent, Long-Acting, Orally Active Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 Diabetes. J. Med. Chem. 2005, 48 (15), 5025– 5037, DOI: 10.1021/jm050261p[ACS Full Text], [CAS], Google Scholar206https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXlsVeltb0%253D&md5=878355880133bb0ddd6bd14ecbf1d070Discovery and Preclinical Profile of Saxagliptin (BMS-477118): A Highly Potent, Long-Acting, Orally Active Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 DiabetesAugeri, David J.; Robl, Jeffrey A.; Betebenner, David A.; Magnin, David R.; Khanna, Ashish; Robertson, James G.; Wang, Aiying; Simpkins, Ligaya M.; Taunk, Prakash; Huang, Qi; Han, Song-Ping; Abboa-Offei, Benoni; Cap, Michael; Xin, Li; Tao, Li; Tozzo, Effie; Welzel, Gustav E.; Egan, Donald M.; Marcinkeviciene, Jovita; Chang, Shu Y.; Biller, Scott A.; Kirby, Mark S.; Parker, Rex A.; Hamann, Lawrence G.Journal of Medicinal Chemistry (2005), 48 (15), 5025-5037CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Efforts to further elucidate structure-activity relationships (SAR) within the authors previously disclosed series of β-quaternary amino acid linked L-cis-4,5-methanoprolinenitrile dipeptidyl peptidase IV (DPP-IV) inhibitors led to the investigation of vinyl substitution at the β-position of α-cycloalkyl-substituted glycines. Despite poor systemic exposure, vinyl-substituted compds. showed extended duration of action in acute rat ex vivo plasma DPP-IV inhibition models. Oxygenated putative metabolites were prepd. and were shown to exhibit the potency and extended duration of action of their precursors in efficacy models measuring glucose clearance in Zuckerfa/fa rats. Extension of this approach to adamantylglycine-derived inhibitors led to the discovery of highly potent inhibitors, including hydroxyadamantyl compd. BMS-477118 (saxagliptin), a highly efficacious, stable, and long-acting DPP-IV inhibitor, which is currently undergoing clin. trials for treatment of type 2 diabetes.
- 207Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C. Nitrile-Containing Pharmaceuticals: Efficacious Roles of the Nitrile Pharmacophore. J. Med. Chem. 2010, 53 (22), 7902– 7917, DOI: 10.1021/jm100762r[ACS Full Text], [CAS], Google Scholar207https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtV2kurvP&md5=040bbaebd9e8c9999d195b9a22221feaNitrile-Containing Pharmaceuticals: Efficacious Roles of the Nitrile PharmacophoreFleming, Fraser F.; Yao, Lihua; Ravikumar, P. C.; Funk, Lee; Shook, Brian C.Journal of Medicinal Chemistry (2010), 53 (22), 7902-7917CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. The review is structured according to the nature of the nitrile-bearing substituent. As the no. of nitrile-contg. drug leads is vast, the review has focused on launched nitrile-contg. pharmaceuticals and currently active clin. candidates. Most nitrile-contg. pharmaceuticals are aroms. with aliph.-, alkene-, and nitrogen-bearing nitriles being progressively less frequent. Within each class, the bioactive nitriles are collated according to common structural elements and mode of action. The hope is that this survey will allow greater deployment of this versatile functionality within drug leads.
- 208Oballa, R. M.; Truchon, J.-F.; Bayly, C. I.; Chauret, N.; Day, S.; Crane, S.; Berthelette, C. A Generally Applicable Method for Assessing the Electrophilicity and Reactivity of Diverse Nitrile-Containing Compounds. Bioorg. Med. Chem. Lett. 2007, 17 (4), 998– 1002, DOI: 10.1016/j.bmcl.2006.11.044[Crossref], [PubMed], [CAS], Google Scholar208https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht12qtr4%253D&md5=b8fff00bdd6a746753f8f244d25b0cc8A generally applicable method for assessing the electrophilicity and reactivity of diverse nitrile-containing compoundsOballa, Renata M.; Truchon, Jean-Francois; Bayly, Christopher I.; Chauret, Nathalie; Day, Stephen; Crane, Sheldon; Berthelette, CarlBioorganic & Medicinal Chemistry Letters (2007), 17 (4), 998-1002CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)Nitrile-based inhibitors of cathepsin K have been known for some time and mechanism-of-action studies have demonstrated that cysteinyl proteases interact with nitriles in a reversible fashion. Three main classes of nitrile-contg. inhibitors have been published in the cathepsin K field: (i) cyanamides, (ii) arom. nitriles, and (iii) aminoacetonitriles. A computational approach was used to calc. the theor. reactivities of diverse nitriles and this was found to correlate with their extent of reactivity with free cysteine. Moreover, there is a tentative link between high reactivity with cysteine and the potential to lead to irreversible covalent binding to proteins.
- 209Schmitz, J.; Beckmann, A.-M.; Dudic, A.; Li, T.; Sellier, R.; Bartz, U.; Gütschow, M. 3-Cyano-3-Aza-β-Amino Acid Derivatives as Inhibitors of Human Cysteine Cathepsins. ACS Med. Chem. Lett. 2014, 5 (10), 1076– 1081, DOI: 10.1021/ml500238q[ACS Full Text], [CAS], Google Scholar209https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlalu7rL&md5=2c500ebf40c879cfbd905fd57ba006863-Cyano-3-aza-β-amino Acid Derivatives as Inhibitors of Human Cysteine CathepsinsSchmitz, Janina; Beckmann, Anna-Madeleine; Dudic, Adela; Li, Tianwei; Sellier, Robert; Bartz, Ulrike; Guetschow, MichaelACS Medicinal Chemistry Letters (2014), 5 (10), 1076-1081CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Nitrile-type inhibitors are known to interact with cysteine proteases in a covalent-reversible manner. The chemotype of 3-cyano-3-aza-β-amino acid derivs. I [R1 = H, Me; R2 = Me, Bn; R3 = Bn; R2R3 = 4-morpholinyl] was designed in which the N-cyano group is centrally arranged in the mol. to allow for interactions with the nonprimed and primed binding regions of the target enzymes. These compds. were evaluated as inhibitors of the human cysteine cathepsins K, S, B, and L. They exhibited slow-binding behavior and were found to be exceptionally potent, in particular toward cathepsin K, with second-order rate consts. up to 52 900 × 103 M-1s-1.
- 210Cai, J.; Fradera, X.; van Zeeland, M.; Dempster, M.; Cameron, K. S.; Bennett, D. J.; Robinson, J.; Popplestone, L.; Baugh, M.; Westwood, P.; Bruin, J.; Hamilton, W.; Kinghorn, E.; Long, C.; Uitdehaag, J. C. M. 4-(3-Trifluoromethylphenyl)-Pyrimidine-2-Carbonitrile as Cathepsin S Inhibitors: N3, Not N1 Is Critically Important. Bioorg. Med. Chem. Lett. 2010, 20 (15), 4507– 4510, DOI: 10.1016/j.bmcl.2010.06.043[Crossref], [PubMed], [CAS], Google Scholar210https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXptVSktL8%253D&md5=c4f3a722e29616dca5e47b0c8cbcadef4-(3-Trifluoromethylphenyl)-pyrimidine-2-carbonitrile as cathepsin S inhibitors: N3, not N1 is critically importantCai, Jiaqiang; Fradera, Xavier; van Zeeland, Mario; Dempster, Maureen; Cameron, Kenneth S.; Bennett, D. Jonathan; Robinson, John; Popplestone, Lucy; Baugh, Mark; Westwood, Paul; Bruin, John; Hamilton, William; Kinghorn, Emma; Long, Clive; Uitdehaag, Joost C. M.Bioorganic & Medicinal Chemistry Letters (2010), 20 (15), 4507-4510CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Using computer aided modeling studies, a new extended P2/S2 interaction was identified. This extended region can accommodate a variety of functional groups, such as aryls and basic amines. It was discovered that the N3 nitrogen of the pyrimidine-2-carbonitrile is crit. for its cathepsin cysteine protease inhibition. N1 nitrogen also contributes to the inhibitory activity, but to a very limited degree. An in situ double activation' mechanism was proposed to explain these results.
- 211Mac Sweeney, A.; Grosche, P.; Ellis, D.; Combrink, K.; Erbel, P.; Hughes, N.; Sirockin, F.; Melkko, S.; Bernardi, A.; Ramage, P.; Jarousse, N.; Altmann, E. Discovery and Structure-Based Optimization of Adenain Inhibitors. ACS Med. Chem. Lett. 2014, 5 (8), 937– 941, DOI: 10.1021/ml500224t[ACS Full Text], [CAS], Google Scholar211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M%252FktFWmsg%253D%253D&md5=d3de233ac94209da27bcef375970a33cDiscovery and structure-based optimization of adenain inhibitorsMac Sweeney Aengus; Grosche Philipp; Erbel Paul; Hughes Nicola; Sirockin Finton; Melkko Samu; Bernardi Anna; Ramage Paul; Altmann Eva; Ellis David; Combrink Keith; Jarousse NadineACS medicinal chemistry letters (2014), 5 (8), 937-41 ISSN:1948-5875.The cysteine protease adenain is the essential protease of adenovirus and, as such, represents a promising target for the treatment of ocular and other adenoviral infections. Through a concise two-pronged hit discovery approach we identified tetrapeptide nitrile 1 and pyrimidine nitrile 2 as complementary starting points for adenain inhibition. These hits enabled the first high-resolution X-ray cocrystal structures of adenain with inhibitors bound and revealed the binding mode of 1 and 2. The screening hits were optimized by a structure-guided medicinal chemistry strategy into low nanomolar drug-like inhibitors of adenain.
- 212de Jesus Cortez, F.; Nguyen, P.; Truillet, C.; Tian, B.; Kuchenbecker, K. M.; Evans, M. J.; Webb, P.; Jacobson, M. P.; Fletterick, R. J.; England, P. M. Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogen. ACS Chem. Biol. 2017, 12 (12), 2934– 2939, DOI: 10.1021/acschembio.7b00702[ACS Full Text], [CAS], Google Scholar212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1Sksr%252FP&md5=3871dfbf0cd53790a8e63f84a1157416Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogende Jesus Cortez, Felipe; Nguyen, Phuong; Truillet, Charles; Tian, Boxue; Kuchenbecker, Kristopher M.; Evans, Michael J.; Webb, Paul; Jacobson, Matthew P.; Fletterick, Robert J.; England, Pamela M.ACS Chemical Biology (2017), 12 (12), 2934-2939CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Resistance to clin. antiandrogens has plagued the evolution of effective therapeutics for advanced prostate cancer. As with the first-line therapeutic bicalutamide (Casodex), resistance to newer antiandrogens (enzalutamide, ARN-509) develops quickly in patients, despite the fact that these drugs have ∼10-fold better affinity for androgen receptor than bicalutamide. Improving affinity alone is often not sufficient to prevent resistance and alternative strategies are needed to improve antiandrogen efficacy. Covalent and reversible covalent drugs are being used to thwart drug resistance in other contexts, and activated aryl nitriles are among the moieties being exploited for this purpose. The authors capitalized on the presence of an aryl nitrile in bicalutamide, and the existence of a native cysteine residue (Cys784) in the androgen receptor ligand binding pocket, to develop 5N-bicalutamide I, a cysteine-reactive antiandrogen. I exhibits a 150-fold improvement in Ki and 20-fold improvement in IC50 over the parent compd. The authors attribute the marked improvement in affinity and activity to the formation of a covalent adduct with Cys784, a residue that is not among the more than 160 androgen receptor point mutations assocd. with prostate cancer. Increasing the residence time of bound antiandrogen via formation of a covalent adduct may forestall the drug resistance seen with current clin. antiandrogens.
- 213Deaton, D. N.; Hassell, A. M.; McFadyen, R. B.; Miller, A. B.; Miller, L. R.; Shewchuk, L. M.; Tavares, F. X.; Willard, D. H.; Wright, L. L. Novel and Potent Cyclic Cyanamide-Based Cathepsin K Inhibitors. Bioorg. Med. Chem. Lett. 2005, 15 (7), 1815– 1819, DOI: 10.1016/j.bmcl.2005.02.033[Crossref], [PubMed], [CAS], Google Scholar213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisVCntL8%253D&md5=7d41d58899ce26c59a839a041149cd53Novel and potent cyclic cyanamide-based cathepsin K inhibitorsDeaton, David N.; Hassell, Anne M.; McFadyen, Robert B.; Miller, Aaron B.; Miller, Larry R.; Shewchuk, Lisa M.; Tavares, Francis X.; Willard, Derril H.; Wright, Lois L.Bioorganic & Medicinal Chemistry Letters (2005), 15 (7), 1815-1819CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Starting from a PDE IV inhibitor hit derived from high throughput screening of the compd. collection, a key pyrrolidine cyanamide pharmacophore was identified. Modifications of the pyrrolidine ring produced enhancements in cathepsin K inhibition. An X-ray co-crystal structure of a cyanamide with cathepsin K confirmed the mode of inhibition.
- 214Falgueyret, J.-P.; Oballa, R. M.; Okamoto, O.; Wesolowski, G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.; Riendeau, D.; Rodan, S. B.; Percival, M. D. Novel, Nonpeptidic Cyanamides as Potent and Reversible Inhibitors of Human Cathepsins K and L. J. Med. Chem. 2001, 44 (1), 94– 104, DOI: 10.1021/jm0003440[ACS Full Text], [CAS], Google Scholar214https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXos1ajs7g%253D&md5=aed27c1353797e413890ea02d900095bNovel, Nonpeptidic Cyanamides as Potent and Reversible Inhibitors of Human Cathepsins K and LFalgueyret, Jean-Pierre; Oballa, Renata M.; Okamoto, Osamu; Wesolowski, Gregg; Aubin, Yves; Rydzewski, Robert M.; Prasit, Peppi; Riendeau, Denis; Rodan, Sevgi B.; Percival, M. DavidJournal of Medicinal Chemistry (2001), 44 (1), 94-104CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Compds. contg. a 1-cyanopyrrolidinyl ring were identified as potent and reversible inhibitors of cathepsins K and L. The original lead compd. I inhibits cathepsins K and L with IC50 values of 0.37 and 0.45 μM, resp. Modification of compd. I by replacement of the quinoline moiety led to the synthesis of N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide (2). Compd. II was found to be a potent inhibitor of cathepsins K and L with a Ki value of 50 nM for cathepsin K. Replacement of the 1-cyanopyrrolidine of compd. II by a 1-cyanoazetidine increased the potency of the inhibitor by 10-fold. This increase in potency is probably due to an enhanced chem. reactivity of the compd. toward the thiolate of the active site of the enzyme. This is demonstrated when the assay is performed in the presence of glutathione at pH 7.0 which favors the formation of a GSH thiolate anion. Under these assay conditions, there is a loss of potency in the 1-cyanoazetidine series due to the formation of an inactive complex between the GSH thiolate and the 1-cyanoazetidine inhibitors. 1-Cyanopyrrolidinyl inhibitors exhibited time-dependent inhibition which allowed us to det. the assocn. and dissocn. rate consts. with human cathepsin K. The kinetic data obtained showed that the increase of potency obsd. between different 1-cyanopyrrolidinyl inhibitors is due to an increase of kon values and that the assocn. of the compd. with the enzyme fits an apparent one-step mechanism. 13C NMR expts. performed with the enzyme papain showed that compd. 2 forms a covalent isothiourea ester adduct with the enzyme. As predicted by the kinetic anal., the addn. of the irreversible inhibitor E64 to the enzyme-cyanopyrrolidinyl complex totally abolished the signal of the isothiourea bond as obsd. by 13C NMR, thereby demonstrating that the formation of the covalent bond with the active site cysteine residue is reversible. Finally, compd. II inhibits bone resorption in an in vitro assay involving rabbit osteoclasts and bovine bone with an IC50 value of 0.7 μM. 1-Cyanopyrrolidine represents a new class of nonpeptidic compds. that inhibit cathepsin K and L activity and proteolysis of bone collagen.
- 215Rydzewski, R. M.; Bryant, C.; Oballa, R.; Wesolowski, G.; Rodan, S. B.; Bass, K. E.; Wong, D. H. Peptidic 1-Cyanopyrrolidines: Synthesis and SAR of a Series of Potent, Selective Cathepsin Inhibitors. Bioorg. Med. Chem. 2002, 10 (10), 3277– 3284, DOI: 10.1016/S0968-0896(02)00173-6[Crossref], [PubMed], [CAS], Google Scholar215https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XlvVems7o%253D&md5=4e40001c5fe70cbf3cb309bb8333bb5dPeptidic 1-cyanopyrrolidines: synthesis and SAR of a series of potent, selective cathepsin inhibitorsRydzewski, Robert M.; Bryant, Clifford; Oballa, Renata; Wesolowski, Gregg; Rodan, Sevgi B.; Bass, Kathryn E.; Wong, Darren H.Bioorganic & Medicinal Chemistry (2002), 10 (10), 3277-3284CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)1-Cyanopyrrolidines have previously been reported to inhibit cysteinyl cathepsins (Falgueyret, J.-P. et al., J. Med. Chem. 2001, 44, 94). To optimize binding interactions for a given cathepsin and simultaneously reduce interactions with the other closely related enzymes, small peptidic substituents were introduced to the 1-cyanopyrrolidine scaffold, either at the 2-position starting with proline or at the 3-position of aminopyrrolidines. The resulting novel compds. proved to be micromolar inhibitors of cathepsin B (Cat B) but nanomolar to picomolar inhibitors of cathepsins K, L, and S (Cat K, Cat L, Cat S). Several of the compds. were >20-fold selective vs. the other 3 cathepsins. SAR trends were obsd., most notably the remarkable potency of Cat L inhibitors based on the 1-cyano-d-proline scaffold. The selectivity of one such compd., the 94 picomolar Cat L inhibitor (I), was demonstrated at higher concns. in DLD-1 cells. Although none of the compds. in the proline series that was tested proved to be submicromolar in the in vitro bone resorption assay, 2 Cat K inhibitors in the 3-substituted pyrrolidine series were relatively potent in that assay.
- 216Lainé, D.; Palovich, M.; McCleland, B.; Petitjean, E.; Delhom, I.; Xie, H.; Deng, J.; Lin, G.; Davis, R.; Jolit, A.; Nevins, N.; Zhao, B.; Villa, J.; Schneck, J.; McDevitt, P.; Midgett, R.; Kmett, C.; Umbrecht, S.; Peck, B.; Davis, A. B.; Bettoun, D. Discovery of Novel Cyanamide-Based Inhibitors of Cathepsin C. ACS Med. Chem. Lett. 2011, 2 (2), 142– 147, DOI: 10.1021/ml100212k[ACS Full Text], [CAS], Google Scholar216https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtl2rsL%252FK&md5=5b8478f6abc28e4d273eb9aa7c93aeb1Discovery of novel cyanamide-based inhibitors of cathepsin CLaine, Dramane; Palovich, Michael; McCleland, Brent; Petitjean, Emilie; Delhom, Isabelle; Xie, Haibo; Deng, Jianghe; Lin, Guoliang; Davis, Roderick; Jolit, Anais; Nevins, Neysa; Zhao, Baoguang; Villa, Jim; Schneck, Jessica; McDevitt, Patrick; Midgett, Robert; Kmett, Casey; Umbrecht, Sandra; Peck, Brian; Davis, Alicia Bacon; Bettoun, DavidACS Medicinal Chemistry Letters (2011), 2 (2), 142-147CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)The discovery of potent and selective cyanamide-based inhibitors of the cysteine protease cathepsin C is detailed. Optimization of the template with regard to plasma stability led to the identification of (2S,4R)-1-cyano-2-methyl-4-(2,5-dibromophenylsulfonylamino)pyrrolidine (I), a potent cathepsin C inhibitor with excellent selectivity over other cathepsins and potent in vivo activity in a cigarette smoke mouse model.
- 217Hill, S. V.; Williams, A.; Longridge, J. L. Acid-Catalysed Hydrolysis of Cyanamides: Estimates of Carbodi-Imide Basicity and Tautomeric Equilibrium Constant between Carbodi-Imide and Cyanamide. J. Chem. Soc., Perkin Trans. 2 1984, 0 (6), 1009– 1013, DOI: 10.1039/p29840001009[Crossref], [CAS], Google Scholar217https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXlslSgsL8%253D&md5=e142ad1a3c0bca71daf9eec01035e538Acid-catalyzed hydrolysis of cyanamides: estimates of carbodiimide basicity and tautomeric equilibrium constant between carbodiimide and cyanamideHill, Stephen V.; Williams, Andrew; Longridge, Jethro L.Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1984), (6), 1009-13CODEN: JCPKBH; ISSN:0300-9580.N-Cyanourea is an intermediate in the hydrolysis of NCNHCN (I), which is shown to react as its carbodiimide tautomer. The hydrolysis of I, H2NCONHCN, and (MeNH)2CNCN (II) is specific acid-catalyzed. General acid catalysis is demonstrated for the hydrolysis of II which is considered to be specific acid-nucleophilic. Assuming the carbodiimide mechanism also holds for the specific acid-catalyzed hydrolysis of H2NCN, the tautomeric equil. const. for formation of the parent carbodiimide in H2O at 25° is 0.6 × 10-7 and the pK of the protonated carbodiimide is estd. to be -1 to +1. Rate consts. are reported for alk. hydrolysis of cyanamides.
- 218Benson, M. J.; Rodriguez, V.; von Schack, D.; Keegan, S.; Cook, T. A.; Edmonds, J.; Benoit, S.; Seth, N.; Du, S.; Messing, D.; Nickerson-Nutter, C. L.; Dunussi-Joannopoulos, K.; Rankin, A. L.; Ruzek, M.; Schnute, M. E.; Douhan, J. Modeling the Clinical Phenotype of BTK Inhibition in the Mature Murine Immune System. J. Immunol. 2014, 193 (1), 185, DOI: 10.4049/jimmunol.1302570[Crossref], [PubMed], [CAS], Google Scholar218https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVWmsLbJ&md5=817796f74f02afc4171dbb6b19368528Modeling the Clinical Phenotype of BTK Inhibition in the Mature Murine Immune SystemBenson, Micah J.; Rodriguez, Varenka; von Schack, David; Keegan, Sean; Cook, Tim A.; Edmonds, Jason; Benoit, Stephen; Seth, Nilufer; Du, Sarah; Messing, Dean; Nickerson-Nutter, Cheryl L.; Dunussi-Joannopoulos, Kyri; Rankin, Andrew L.; Ruzek, Melanie; Schnute, Mark E.; Douhan, JohnJournal of Immunology (2014), 193 (1), 185-197CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncol. and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clin. phenotype of BTK inhibition by systematically examg. the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clin. phenotype of chronic BTK inhibition.
- 219Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.; Almaden, C.; Nagata, A.; Ryan, K.; Feng, J.; Dalvie, D.; Kath, J. C.; Xu, M.; Wani, R.; Murray, B. W. Covalent EGFR Inhibitor Analysis Reveals Importance of Reversible Interactions to Potency and Mechanisms of Drug Resistance. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (1), 173– 178, DOI: 10.1073/pnas.1313733111[Crossref], [PubMed], [CAS], Google Scholar219https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXps1eluw%253D%253D&md5=47197c5e7876a238eefb2e8e7de305eaCovalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistanceSchwartz, Phillip A.; Kuzmic, Petr; Solowiej, James; Bergqvist, Simon; Bolanos, Ben; Almaden, Chau; Nagata, Asako; Ryan, Kevin; Feng, Junli; Dalvie, Deepak; Kath, John C.; Xu, Meirong; Wani, Revati; Murray, Brion WilliamProceedings of the National Academy of Sciences of the United States of America (2014), 111 (1), 173-178CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chem. reactivity in overall potency are not well-understood. To characterize the underlying mol. processes at a microscopic level and det. the appropriate kinetic consts., specialized exptl. design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 105-107 M-1s-1), despite their low specific reactivity (kinact ≤ 2.1 × 10-3 s-1), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissocn. and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidn. on enzyme catalysis and inhibitor pharmacol. are characterized. Oxidn. of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidn. (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors.
- 220Zapf, C. W.; Gerstenberger, B. S.; Xing, L.; Limburg, D. C.; Anderson, D. R.; Caspers, N.; Han, S.; Aulabaugh, A.; Kurumbail, R.; Shakya, S.; Li, X.; Spaulding, V.; Czerwinski, R. M.; Seth, N.; Medley, Q. G. Covalent Inhibitors of Interleukin-2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood Assay. J. Med. Chem. 2012, 55 (22), 10047– 10063, DOI: 10.1021/jm301190s[ACS Full Text], [CAS], Google Scholar220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFOlsbnP&md5=7e39a17151dc9493d13439bc48fa83a3Covalent Inhibitors of Interleukin-2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood AssayZapf, Christoph W.; Gerstenberger, Brian S.; Xing, Li; Limburg, David C.; Anderson, David R.; Caspers, Nicole; Han, Seungil; Aulabaugh, Ann; Kurumbail, Ravi; Shakya, Subarna; Li, Xin; Spaulding, Vikki; Czerwinski, Robert M.; Seth, Nilufer; Medley, Quintus G.Journal of Medicinal Chemistry (2012), 55 (22), 10047-10063CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concns. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogs are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approx. 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.
- 221Gupta, P.; Wright, S. E.; Kim, S.-H.; Srivastava, S. K. Phenethyl Isothiocyanate: A Comprehensive Review of Anti-Cancer Mechanisms. Biochim. Biophys. Acta, Rev. Cancer 2014, 1846 (2), 405– 424, DOI: 10.1016/j.bbcan.2014.08.003[Crossref], [CAS], Google Scholar221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVKnt7fM&md5=ba0ae7ed36d1f72329d7257393f24df7Phenethyl isothiocyanate: A comprehensive review of anti-cancer mechanismsGupta, Parul; Wright, Stephen E.; Kim, Sung-Hoon; Srivastava, Sanjay K.Biochimica et Biophysica Acta, Reviews on Cancer (2014), 1846 (2), 405-424CODEN: BBACEU; ISSN:0304-419X. (Elsevier B.V.)A review. The epidemiol. evidence suggests a strong inverse relationship between dietary intake of cruciferous vegetables and the incidence of cancer. Among other constituents of cruciferous vegetables, isothiocyanates (ITC) are the main bioactive chems. present. Phenethyl isothiocyanate (PEITC) is present as gluconasturtiin in many cruciferous vegetables with remarkable anti-cancer effects. PEITC is known to not only prevent the initiation phase of carcinogenesis process but also to inhibit the progression of tumorigenesis. PEITC targets multiple proteins to suppress various cancer-promoting mechanisms such as cell proliferation, progression and metastasis. Pre-clin. evidence suggests that combination of PEITC with conventional anti-cancer agents is also highly effective in improving overall efficacy. Based on accumulating evidence, PEITC appears to be a promising agent for cancer therapy and is already under clin. trials for leukemia and lung cancer. This is the first review which provides a comprehensive anal. of known targets and mechanisms along with a crit. evaluation of PEITC as a future anti-cancer agent.
- 222Hinman, A.; Chuang, H.; Bautista, D. M.; Julius, D. TRP Channel Activation by Reversible Covalent Modification. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (51), 19564– 19568, DOI: 10.1073/pnas.0609598103[Crossref], [PubMed], [CAS], Google Scholar222https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhsVGksg%253D%253D&md5=3fb76ecf2a1d5d0992ade1873b2d5bccTRP channel activation by reversible covalent modificationHinman, Andrew; Chuang, Huai-hu; Bautista, Diana M.; Julius, DavidProceedings of the National Academy of Sciences of the United States of America (2006), 103 (51), 19564-19568CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Allyl isothiocyanate, the pungent principle of wasabi and other mustard oils, produces pain by activating TRPA1, an excitatory ion channel on sensory nerve endings. Isothiocyanates are membrane--permeable electrophiles that form adducts with thiols and primary amines, suggesting that covalent modification, rather than classical lock-and-key binding, accounts for their agonist properties. Indeed, we show that the thio1 reactive compds. of diverse structure activate TRPA1 in a manner that relies on covalent modification of cysteine residues within the cytoplasmic N terminus of the channel. These findings suggest an unusual paradigm whereby natural products activate a receptor through direct, reversible, and covalent protein modification.
- 223van Bladeren, P. J. Glutathione Conjugation as a Bioactivation Reaction. Chem.-Biol. Interact. 2000, 129 (1), 61– 76, DOI: 10.1016/S0009-2797(00)00214-3[Crossref], [PubMed], [CAS], Google Scholar223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXitlOntA%253D%253D&md5=398ba7a04c67671459a16bc4fe9a09b1Glutathione conjugation as a bioactivation reactionvan Bladeren, P. J.Chemico-Biological Interactions (2000), 129 (1-2), 61-76CODEN: CBINA8; ISSN:0009-2797. (Elsevier Science Ireland Ltd.)A review and discussion with 86 refs. In general, glutathione conjugation is regarded as a detoxication reaction. However, depending on the properties of the substrate, bioactivation is also possible. Four types of activation reaction have been recognized: direct-acting compds., conjugates that are activated through cysteine conjugate beta-lyase, conjugates that are activated through redox cycling and lastly conjugates that release the original reactive parent compd. The glutathione S-transferases have three connections with the formation of bioactivated conjugates: they catalyze their formation in a no. of cases, they are the earliest available target for covalent binding by these conjugates and lastly, the parent alkylating agents are regularly involved in the induction of the enzymes. Individual susceptibility for each of these agents is detd. by individual transferase subunit compn. and methods are becoming available to assess this susceptibility.
- 224Drobnica, L.; Kristián, P.; Augustín, J. The Chemistry of the NCS Group. Cyanates Their Thio Derivatives; Patai, P., Ed.; Wiley: New York, 1977; Part 2, pp 1003– 1221 DOI: 10.1002/9780470771532.ch6 .
- 225Shibata, T.; Kimura, Y.; Mukai, A.; Mori, H.; Ito, S.; Asaka, Y.; Oe, S.; Tanaka, H.; Takahashi, T.; Uchida, K. Transthiocarbamoylation of Proteins by Thiolated Isothiocyanates. J. Biol. Chem. 2011, 286, 42150, DOI: 10.1074/jbc.M111.308049[Crossref], [PubMed], [CAS], Google Scholar225https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFKjsr7E&md5=95fec76a0cce811c4dcfce40e8512ec8Transthiocarbamoylation of Proteins by Thiolated IsothiocyanatesShibata, Takahiro; Kimura, Yuuki; Mukai, Akihiro; Mori, Hitoshi; Ito, Sohei; Asaka, Yukio; Oe, Sho; Tanaka, Hiroshi; Takahashi, Takashi; Uchida, KojiJournal of Biological Chemistry (2011), 286 (49), 42150-42161, S42150/1-S42150/13CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Isothiocyanates, membrane-permeable electrophiles that form adducts with thiols, have been suggested to have important medical benefits. Here we shed light on isothiocyanate-thiol conjugates and studied their electrophilic potential transferring an isothiocyanate moiety to cellular proteins. When we examd. the effect of sulfhydryl mols. on cellular response induced by 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analog of sulforaphane isolated from broccoli, we obsd. significant induction of heme oxygenase-1 by 6-HITC even in the presence of N-acetyl-L-cysteine or glutathione (GSH). In addn., the authentic 6-HITC-β-mercaptoethanol (6-HITC-ME) conjugate markedly up-regulated the enzyme expression, suggesting the electrophilic potential of thiolated isothiocyanates. To gain a chem. insight into the cellular response induced by thiolated isothiocyanates, we studied the occurrence of transthiocarbamoylation of sulfhydryl mols. by 6-HITC-ME and obsd. that, upon incubation of 6-HITC-ME with GSH, a single product corresponding to the GSH conjugate of 6-HITC was generated. To test the functional ability of thiolated isothiocyanates to thiocarbamoylate proteins in living cells, we designed a novel probe, combining an isothiocyanate-reactive group and an alkyne functionality, and revealed that the transthiocarbamoylation of proteins occurred in the cells upon exposure to 6-HITC-ME. The target of thiocarbamoylation included heat shock protein 90 β (Hsp90β), a chaperone ATPase of the Hsp90 family implicated in protein maturation and targeting. To identify the sites of the Hsp90β modification, we utilized nano-LC/MALDI-TOF MS/MS and suggested that a thiol group on the peptide contg. Cys-521 reacted with 6-HITC, resulting in a covalent adduct in a 6-HITC-treated recombinant Hsp90β in vitro. The site-selective binding to Cys-521 was supported by in silico modeling. Further study on the thiocarbamoylation of Hsp90β suggested that the formation of 6-HITC-Hsp90β conjugate might cause activation of heat shock factor-1, rapidly signaling a potential heat shock response. These data suggest that thiolated isothiocyanates are an active metabolite that could contribute to cellular responses through transthiocarbamoylation of cellular proteins.
- 226Nakamura, T.; Kawai, Y.; Kitamoto, N.; Osawa, T.; Kato, Y. Covalent Modification of Lysine Residues by Allyl Isothiocyanate in Physiological Conditions: Plausible Transformation of Isothiocyanate from Thiol to Amine. Chem. Res. Toxicol. 2009, 22 (3), 536– 542, DOI: 10.1021/tx8003906[ACS Full Text], [CAS], Google Scholar226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhvFGms7Y%253D&md5=19f9ae27e52e9c9bfde9c1e27cabac62Covalent Modification of Lysine Residues by Allyl Isothiocyanate in Physiological Conditions: Plausible Transformation of Isothiocyanate from Thiol to AmineNakamura, Toshiyuki; Kawai, Yoshichika; Kitamoto, Noritoshi; Osawa, Toshihiko; Kato, YojiChemical Research in Toxicology (2009), 22 (3), 536-542CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)We investigated the reactivity of allyl isothiocyanate (AITC) with amino groups under physiol. conditions. First, the chem. reaction of AITC with bovine serum albumin (BSA) was investigated. When BSA was incubated with AITC in a phosphate buffer (pH 7.4), the loss of Lys residues was obsd. Second, the Lys residue Nα-benzoyl-glycyl-L-lysine (BGK) was reacted with AITC in the buffer, and a novel peak was detected using high performance liq. chromatog. (HPLC). The peak was purified and identified as AITC-modified BGK with a N.vepsiln.-thiocarbamoyl linkage. However, a thiol residue is known to be a predominant target of an isothiocyanate (ITC). Although AITC may react with a thiol moiety in vivo, a thiocarbamoyl linkage between ITC and thiol is unstable, and an AITC mol. may be regenerated. To prove the plausible transformation of ITC from thiol to amine, synthetic AITC-conjugated Nα-acetyl-L-cysteine (NAC) was incubated with BGK at 37° in physiol. buffer, and the generation of AITC-Lys was analyzed. The loss of the AITC-NAC adduct corresponded to the formation of the AITC-BGK adduct. Furthermore, using a novel monoclonal antibody (A4C7mAb) specific for AITC-Lys, we found that the AITC-Lys residue was generated from the reaction between AITC-NAC and BSA. Although AITC preferentially reacts with thiol rather than with Lys, AITC can be liberated from thiols and can then react with amino groups. The ITC-Lys adduct may be a useful marker for ITC target mols.
- 227Kumari, V.; Dyba, M. A.; Holland, R. J.; Liang, Y.-H.; Singh, S. V.; Ji, X. Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical. PLoS One 2016, 11 (9), e0163821, DOI: 10.1371/journal.pone.0163821[Crossref], [PubMed], [CAS], Google Scholar227https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitFGqtrY%253D&md5=3e02a10dd6bb210a738c0cb61d2bc48fIrreversible inhibition of glutathione S-transferase by phenethyl isothiocyanate (PEITC), a dietary cancer chemopreventive phytochemicalKumari, Vandana; Dyba, Marzena A.; Holland, Ryan J.; Liang, Yu-He; Singh, Shivendra V.; Ji, XinhuaPLoS One (2016), 11 (9), e0163821/1-e0163821/12CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Dietary isothiocyanates abundant as glucosinolate precursors in many edible cruciferous vegetables are effective for prevention of cancer in chem.-induced and transgenic rodent models. Some of these agents, including phenethyl isothiocyanate (PEITC), have already advanced to clin. investigations. The primary route of isothiocyanate metab. is its conjugation with glutathione (GSH), a reaction catalyzed by glutathione S-transferase (GST). The pi class GST of subunit type 1 (hGSTP1) is much more effective than the alpha class GST of subunit type 1 (hGSTA1) in catalyzing the conjugation. Here, we report the crystal structures of hGSTP1 and hGSTA1 each in complex with the GSH adduct of PEITC. We find that PEITC also covalently modifies the cysteine side chains of GST, which irreversibly inhibits enzymic activity.
- 228Wilson, A. J.; Kerns, J. K.; Callahan, J. F.; Moody, C. J. Keap Calm, and Carry on Covalently. J. Med. Chem. 2013, 56 (19), 7463– 7476, DOI: 10.1021/jm400224q[ACS Full Text], [CAS], Google Scholar228https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVKqtbvE&md5=ea907fb2ccd8c28142ab4fa2776d0195Keap Calm, and Carry on CovalentlyWilson, Anthony J.; Kerns, Jeffrey K.; Callahan, James F.; Moody, Christopher J.Journal of Medicinal Chemistry (2013), 56 (19), 7463-7476CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. The Nrf2-Keap1 system plays a major role in cellular defense against oxidative stress. Upon exposure to electrophiles, the cysteine-rich protein Keap1 is covalently modified, and it is this modification of Keap1 that allows the accumulation and subsequent nuclear translocation of Nrf2 where it induces the transcription of over 100 protective genes. This mechanism can be exploited in drug discovery approaches to diseases such as chronic kidney disease (CKD), chronic obstructive pulmonary disease (COPD), asthma, and neurodegenerative diseases like multiple sclerosis (MS) and Parkinson's, utilizing the modification of Keap1 by electrophiles, compds. that would not normally be considered useful in drug discovery programs. This Perspective discusses the development of potential therapies based on potent electrophiles, such as isothiocyanates and Michael acceptors, that, far from being assocd. with toxic events, can actually initiate a range of beneficial protective pathways.
- 229Lewis, S. M.; Li, Y.; Catalano, M. J.; Laciak, A. R.; Singh, H.; Seiner, D. R.; Reilly, T. J.; Tanner, J. J.; Gates, K. S. Inactivation of Protein Tyrosine Phosphatases by Dietary Isothiocyanates. Bioorg. Med. Chem. Lett. 2015, 25 (20), 4549– 4552, DOI: 10.1016/j.bmcl.2015.08.065[Crossref], [PubMed], [CAS], Google Scholar229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVCnu7vF&md5=aaeef7137d097799f44c0d5b1030e709Inactivation of protein tyrosine phosphatases by dietary isothiocyanatesLewis, Sarah M.; Li, Ya; Catalano, Michael J.; Laciak, Adrian R.; Singh, Harkewal; Seiner, Derrick R.; Reilly, Thomas J.; Tanner, John J.; Gates, Kent S.Bioorganic & Medicinal Chemistry Letters (2015), 25 (20), 4549-4552CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Isothiocyanates are bioactive dietary phytochems. that react readily with protein thiol groups. We find that isothiocyanates are time-dependent inactivators of cysteine-dependent protein tyrosine phosphatases (PTPs). Rate consts. for the inactivation of PTP1B and SHP-2 by allyl isothiocyanate (AITC) and sulforaphane (SF) range from 2 to 16 M-1 s-1. Results in the context of PTP1B are consistent with a mechanism involving covalent, yet reversible, modification of the enzyme's active site cysteine residue.
- 230Cross, J. V.; Foss, F. W.; Rady, J. M.; Macdonald, T. L.; Templeton, D. J. The Isothiocyanate Class of Bioactive Nutrients Covalently Inhibit the MEKK1 Protein Kinase. BMC Cancer 2007, 7 (1), 183, DOI: 10.1186/1471-2407-7-183[Crossref], [PubMed], [CAS], Google Scholar230https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2snmvFKjug%253D%253D&md5=e17c3a1681930fa43ed36a5e1d5f72d3The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinaseCross Janet V; Foss Frank W; Rady Joshua M; Macdonald Timothy L; Templeton Dennis JBMC cancer (2007), 7 (), 183 ISSN:.BACKGROUND: Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. METHODS: The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. RESULTS: ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. CONCLUSION: These results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins.
- 231Mi, L.; Xiao, Z.; Hood, B. L.; Dakshanamurthy, S.; Wang, X.; Govind, S.; Conrads, T. P.; Veenstra, T. D.; Chung, F.-L. Covalent Binding to Tubulin by Isothiocyanates: A Mechanism of Cell Growth Arrest and Apoptosis. J. Biol. Chem. 2008, 283 (32), 22136– 22146, DOI: 10.1074/jbc.M802330200[Crossref], [PubMed], [CAS], Google Scholar231https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXpt1artLc%253D&md5=de1c9ebf6ac3fbb4631e2cd26bbf955eCovalent Binding to Tubulin by Isothiocyanates: a mechanism of cell growth arrest and apoptosisMi, Lixin; Xiao, Zhen; Hood, Brian L.; Dakshanamurthy, Sivanesan; Wang, Xiantao; Govind, Sudha; Conrads, Thomas P.; Veenstra, Timothy D.; Chung, Fung-LungJournal of Biological Chemistry (2008), 283 (32), 22136-22146CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochem. mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and mol. mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examd. whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymn. in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.
- 232Ouertatani-Sakouhi, H.; El-Turk, F.; Fauvet, B.; Roger, T.; Le Roy, D.; Karpinar, D. P.; Leng, L.; Bucala, R.; Zweckstetter, M.; Calandra, T.; Lashuel, H. A. A New Class of Isothiocyanate-Based Irreversible Inhibitors of Macrophage Migration Inhibitory Factor. Biochemistry 2009, 48 (41), 9858– 9870, DOI: 10.1021/bi900957e[ACS Full Text], [CAS], Google Scholar232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFOksrfJ&md5=740dd080eca41baa8b21d8f71e02682aA New Class of Isothiocyanate-Based Irreversible Inhibitors of Macrophage Migration Inhibitory FactorOuertatani-Sakouhi, Hajer; El-Turk, Farah; Fauvet, Bruno; Roger, Thierry; Le Roy, Didier; Karpinar, Damla Pinar; Leng, Lin; Bucala, Richard; Zweckstetter, Markus; Calandra, Thierry; Lashuel, Hilal A.Biochemistry (2009), 48 (41), 9858-9870CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biol. activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogs occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochem., biophys., and structural studies to det. the effect of BITC and its analogs on the conformational state, quaternary structure, catalytic activity, receptor binding, and biol. activity of MIF. Light scattering, anal. ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodn. stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a redn. in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochem. and biol. activities of MIF and present new opportunities for modulating MIF biol. activities in vivo.
- 233Brown, K. K.; Hampton, M. B. Biological Targets of Isothiocyanates. Biochim. Biophys. Acta, Gen. Subj. 2011, 1810 (9), 888– 894, DOI: 10.1016/j.bbagen.2011.06.004[Crossref], [PubMed], [CAS], Google Scholar233https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptlantrs%253D&md5=1d34ab0921044fe7e7aa0eee3622e964Biological targets of isothiocyanatesBrown, Kristin K.; Hampton, Mark B.Biochimica et Biophysica Acta, General Subjects (2011), 1810 (9), 888-894CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)A review. Isothiocyanates are phytochems. with a broad array of effects in biol. systems. Bioactivity includes the stimulation of cellular antioxidant systems, induction of apoptosis and interference with cytokine prodn. and activity. Epidemiol. evidence and exptl. studies indicate that naturally occurring isothiocyanates and synthetic derivs. have anti-cancer and anti-inflammatory properties. This review focuses on the mol. targets of isothiocyanates, and how target modification translates into a biol. response. Isothiocyanates may mediate their effects via direct protein modification or indirectly by disruption of redox homeostasis and increased thiol oxidn. Some target proteins have been identified, but in-depth searches with new techniques are needed to reveal novel targets. Site-directed mutagenesis and isothiocyanate structure-activity relationships will assist in detg. the biol. significance of specific modifications. General significance: Target identification is important for rational drug design and exploiting the therapeutic potential of isothiocyanates. It also provides insight into the diverse pathways that these compds. regulate.
- 234Pearson, R. J.; Blake, D. G.; Mezna, M.; Fischer, P. M.; Westwood, N. J.; McInnes, C. The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1. Cell Chem. Biol. 2018, 25, 1107– 1116, DOI: 10.1016/j.chembiol.2018.06.001[Crossref], [PubMed], [CAS], Google Scholar234https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlart7%252FE&md5=29ef6760403e0925cc2c142bc85b0fa8The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1Pearson, Russell J.; Blake, David G.; Mezna, Mokdad; Fischer, Peter M.; Westwood, Nicholas J.; McInnes, CampbellCell Chemical Biology (2018), 25 (9), 1107-1116.e4CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)The polo kinase family are important oncol. targets that act in regulating entry into and progression through mitosis. Structure-guided discovery of a new class of inhibitors of Polo-like kinase 1 (PLK1) catalytic activity that interact with Cys67 of the ATP binding site is described. Compds. contg. the benzothiazole N-oxide scaffold not only bind covalently to this residue, but are reversible inhibitors through the formation of Meisenheimer complexes. This mechanism of kinase inhibition results in compds. that can target PLK1 with high selectivity, while avoiding issues with irreversible covalent binding and interaction with other thiol-contg. mols. in the cell. Due to renewed interest in covalent drugs and the plethora of potential drug targets, these represent prototypes for the design of kinase inhibitory compds. that achieve high specificity through covalent interaction and yet still bind reversibly to the ATP cleft, a strategy that could be applied to avoid issues with conventional covalent binders.
- 235Federici, L.; Lo Sterzo, C.; Pezzola, S.; Di Matteo, A. D.; Scaloni, F.; Federici, G.; Caccuri, A. M. Structural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-Transferases. Cancer Res. 2009, 69 (20), 8025– 8034, DOI: 10.1158/0008-5472.CAN-09-1314[Crossref], [PubMed], [CAS], Google Scholar235https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1ynurfN&md5=2ead5fbde7543b8a7983908ceedaf57eStructural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-TransferasesFederici, Luca; Lo Sterzo, Carlo; Pezzola, Silvia; Di Matteo, Adele; Scaloni, Flavio; Federici, Giorgio; Caccuri, Anna MariaCancer Research (2009), 69 (20), 8025-8034CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)Glutathione S-transferases (GST) constitute a superfamily of enzymes with diversified functions including detoxification from xenobiotics. In many human cancers, Pi class GST (GSTP1-1) is overexpressed and contributes to multidrug resistance by conjugating chemotherapeutics. In addn., GSTP1-1 displays antiapoptotic activity by interacting with c-Jun NH2-terminal kinase, a key regulator of apoptosis. Therefore, GSTP1-1 is considered a promising target for pharmaceutical treatment. Recently, a potent inhibitor of GSTs, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), was identified and tested on several tumor cell lines demonstrating high antiproliferative activity. To establish the structural basis of NBDHEX activity, we detd. the crystal structure of NBDHEX bound to either GSTP1-1 or GSTM2-2 (mu class). NBDHEX in both cases binds to the H-site but occupies different positions. Furthermore, the compd. is covalently attached to the GSH sulfur in the GSTM2-2 crystal, forming a σ-complex, although it is bound but not conjugated in the GSTP1-1 crystal. Several differences in the H-sites of the two isoenzymes det. the higher affinity of NBDHEX for GSTM2-2 with respect to GSTP1-1. One such difference is the presence of Ile104 in GSTP1-1 close to the bound NBDHEX, whereas the corresponding position is occupied by an alanine in GSTM2-2. Mutation of Ile104 into valine is a frequent GSTP1-1 polymorphism and we show here that the Ile104Val and Ile104Ala variants display a 4-fold higher affinity for the compd. Remarkably, the GSTP1-1/Ile104Ala structure in complex with NBDHEX shows a considerable shift of the compd. inside the H-site. These data might be useful for the development of new anticancer compds.
- 236Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal, M.; Stroud, R. M.; Gordon, E. M.; Wells, J. A. Site-Directed Ligand Discovery. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (17), 9367– 9372, DOI: 10.1073/pnas.97.17.9367[Crossref], [PubMed], [CAS], Google Scholar236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmtVensr8%253D&md5=fef57a0d21af9bc7f6f1f4bf8c023ae5Site-directed ligand discoveryErlanson, Daniel A.; Braisted, Andrew C.; Raphael, Darren R.; Randal, Mike; Stroud, Robert M.; Gordon, Eric M.; Wells, James A.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (17), 9367-9372CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We report a strategy (called "tethering") to discover low mol. wt. ligands (≈250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-contg. mols. (≈1,200 compds.) at concns. typically used in drug screening (10 to 200 μM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metab. with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (Ki≈1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallog. structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.
- 237Zong, L.; Bartolami, E.; Abegg, D.; Adibekian, A.; Sakai, N.; Matile, S. Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension. ACS Cent. Sci. 2017, 3 (5), 449– 453, DOI: 10.1021/acscentsci.7b00080[ACS Full Text], [CAS], Google Scholar237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsFOrsL4%253D&md5=6f68eab2bcb1b6100fb5970d1ee88a06Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest TensionZong, Lili; Bartolami, Eline; Abegg, Daniel; Adibekian, Alexander; Sakai, Naomi; Matile, StefanACS Central Science (2017), 3 (5), 449-453CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)The disulfide dihedral angle in epidithiodiketopiperazines (ETPs) is near 0°. Application of this highest possible ring tension to strain-promoted thiol-mediated uptake results in efficient delivery to the cytosol and nucleus. Compared to the previous best asparagusic acid (AspA), ring-opening disulfide exchange with ETPs occurs more efficiently even with nonactivated thiols, and the resulting thiols exchange rapidly with nonactivated disulfides. ETP-mediated cellular uptake is more than 20 times more efficient compared to AspA, occurs without endosomal capture, depends on temp., and is "unstoppable" by inhibitors of endocytosis and conventional thiol-mediated uptake, including siRNA against the transferrin receptor. These results suggest that ETP-mediated uptake not only maximizes delivery to the cytosol and nucleus but also opens the door to a new multitarget hopping mode of action.
- 238Tjin, C. C.; Otley, K. D.; Baguley, T. D.; Kurup, P.; Xu, J.; Nairn, A. C.; Lombroso, P. J.; Ellman, J. A. Glutathione-Responsive Selenosulfide Prodrugs as a Platform Strategy for Potent and Selective Mechanism-Based Inhibition of Protein Tyrosine Phosphatases. ACS Cent. Sci. 2017, 3 (12), 1322– 1328, DOI: 10.1021/acscentsci.7b00486[ACS Full Text], [CAS], Google Scholar238https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWnsrfN&md5=a1eff343feab9afb7b79628bf5efa034Glutathione-Responsive Selenosulfide Prodrugs as a Platform Strategy for Potent and Selective Mechanism-Based Inhibition of Protein Tyrosine PhosphatasesTjin, Caroline Chandra; Otley, Kate D.; Baguley, Tyler D.; Kurup, Pradeep; Xu, Jian; Nairn, Angus C.; Lombroso, Paul J.; Ellman, Jonathan A.ACS Central Science (2017), 3 (12), 1322-1328CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Dysregulation of protein tyrosine phosphorylation has been implicated in a no. of human diseases, including cancer, diabetes, and neurodegenerative disease. As a result of their essential role in regulating protein tyrosine phosphorylation levels, protein tyrosine phosphatases (PTPs) have emerged as important yet challenging therapeutic targets. Here the authors report on the development and application of a glutathione-responsive motif to facilitate the efficient intracellular delivery of a novel class of selenosulfide phosphatase inhibitors for the selective active site directed inhibition of the targeted PTP by selenosulfide exchange with the active site cysteine. The strategy leverages the large difference in extracellular and intracellular glutathione levels to deliver selenosulfide phosphatase inhibitors to cells. As an initial exploration of the prodrug platform and the corresponding selenosulfide covalent inhibitor class, potent and selective inhibitors were developed for two therapeutically relevant PTP targets: the Mycobacterium tuberculosis virulence factor mPTPA and the CNS-specific tyrosine phosphatase, STtriatal-Enriched protein tyrosine Phosphatase (STEP). The lead selenosulfide inhibitors enable potent and selective inhibition of their resp. targets over a panel of human PTPs and a representative cysteine protease. Kinetic parameters of the inhibitors were characterized, including reversibility of inhibition and rapid rate of GSH exchange at intracellular GSH concns. Addnl., active site covalent inhibitor-labeling with an mPTPA inhibitor was rigorously confirmed by mass spectrometry and cellular activity was demonstrated with a STEP prodrug inhibitor in cortical neurons.
- 239Weichert, D.; Gmeiner, P. Covalent Molecular Probes for Class A G Protein-Coupled Receptors: Advances and Applications. ACS Chem. Biol. 2015, 10 (6), 1376– 1386, DOI: 10.1021/acschembio.5b00070[ACS Full Text], [CAS], Google Scholar239https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtlamsrw%253D&md5=1defbb0685a38101822c936eac6ba2f1Covalent Molecular Probes for Class A G Protein-Coupled Receptors: Advances and ApplicationsWeichert, Dietmar; Gmeiner, PeterACS Chemical Biology (2015), 10 (6), 1376-1386CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A review. Covalent modification of G protein-coupled receptors (GPCRs) by employing specific mol. probes has for decades provided a successful strategy to facilitate the elucidation of the structure and function of this pharmacol. important class of membrane proteins. The ligands typically comprise a pharmacophore that generates affinity for a given GPCR and contain a reactive functionality that may form a covalent bond with a suitably positioned amino acid residue. Covalent ligands have been successfully applied to circumvent poor affinity of compds. when stable labeling of receptor populations was required, and they have been used in the isolation, purifn., and pharmacol. characterization of specific subtypes of GPCRs. Recently, structural studies have demonstrated that covalent mol. probes are effective at stabilizing GPCRs to obtain X-ray crystal structures, thus providing valuable insights for the development of novel therapeutics. Herein, we review covalently binding mol. probes for class A GPCRs with a focus on ligands comprising crosslinking groups that do not require photoactivation and further highlight their significant and diverse applications.
- 240Rosenbaum, D. M.; Zhang, C.; Lyons, J. A.; Holl, R.; Aragao, D.; Arlow, D. H.; Rasmussen, S. G. F.; Choi, H.-J.; DeVree, B. T.; Sunahara, R. K.; Chae, P. S.; Gellman, S. H.; Dror, R. O.; Shaw, D. E.; Weis, W. I.; Caffrey, M.; Gmeiner, P.; Kobilka, B. K. Structure and Function of an Irreversible Agonist-Β2 Adrenoceptor Complex. Nature 2011, 469 (7329), 236– 240, DOI: 10.1038/nature09665[Crossref], [PubMed], [CAS], Google Scholar240https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkvFehsQ%253D%253D&md5=8629b93e43fb692395e2aa6f8bb011a9Structure and function of an irreversible agonist-β2 adrenoceptor complexRosenbaum, Daniel M.; Zhang, Cheng; Lyons, Joseph A.; Holl, Ralph; Aragao, David; Arlow, Daniel H.; Rasmussen, Soren G. F.; Choi, Hee-Jung; DeVree, Brian T.; Sunahara, Roger K.; Chae, Pil Seok; Gellman, Samuel H.; Dror, Ron O.; Shaw, David E.; Weis, William I.; Caffrey, Martin; Gmeiner, Peter; Kobilka, Brian K.Nature (London, United Kingdom) (2011), 469 (7329), 236-240CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biol. function by initiating cellular signaling in response to chem. diverse agonists. Despite recent progress in the structural biol. of GPCRs, the mol. basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystn. of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β2 adrenergic receptor (β2AR) as a guide, we designed a β2AR agonist that can be covalently tethered to a specific site on the receptor through a disulfide bond. The covalent β2AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystd. a covalent agonist-bound β2AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and detd. its structure at 3.5 Å resoln. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) mol. dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.
- 241Schwalbe, T.; Kaindl, J.; Hübner, H.; Gmeiner, P. Potent Haloperidol Derivatives Covalently Binding to the Dopamine D2 Receptor. Bioorg. Med. Chem. 2017, 25 (19), 5084– 5094, DOI: 10.1016/j.bmc.2017.06.034[Crossref], [PubMed], [CAS], Google Scholar241https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVyntb7O&md5=c7357c0703b5ee0ee9c7ba496fcef4e4Potent haloperidol derivatives covalently binding to the dopamine D2 receptorSchwalbe, Tobias; Kaindl, Jonas; Huebner, Harald; Gmeiner, PeterBioorganic & Medicinal Chemistry (2017), 25 (19), 5084-5094CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)The dopamine D2 receptor (D2R) is a common drug target for the treatment of a variety of neurol. disorders including schizophrenia. Structure based design of subtype selective D2R antagonists requires high resoln. crystal structures of the receptor and pharmacol. tools promoting a better understanding of the protein-ligand interactions. Recently, we reported the development of a chem. activated dopamine deriv. (FAUC150) designed to covalently bind the L94C mutant of the dopamine D2 receptor. Using FAUC150 as a template, we elaborated the design and synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming covalent D2R-ligand complexes. The disulfide- and Michael acceptor-functionalized compds. showed significant receptor affinity and an irreversible binding profile in radioligand depletion expts.
- 242Liu, Y.; Xie, Z.; Zhao, D.; Zhu, J.; Mao, F.; Tang, S.; Xu, H.; Luo, C.; Geng, M.; Huang, M.; Li, J. Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) Inhibitors. J. Med. Chem. 2017, 60 (6), 2227– 2244, DOI: 10.1021/acs.jmedchem.6b01245[ACS Full Text], [CAS], Google Scholar242https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjtlyjtr0%253D&md5=7529ce2fc9d1a138a8d1405a23f7e4e8Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) InhibitorsLiu, Yifu; Xie, Zuoquan; Zhao, Dan; Zhu, Jin; Mao, Fei; Tang, Shuai; Xu, Hui; Luo, Cheng; Geng, Meiyu; Huang, Min; Li, JianJournal of Medicinal Chemistry (2017), 60 (6), 2227-2244CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Pyruvate dehydrogenase kinases (PDKs) are overexpressed in most cancer cells and are responsible for aberrant glucose metab. The authors previously described Bis(4-morpholinyl thiocarbonyl)-disulfide (JX06, 16) as the first covalent inhibitor of PDK1. Here, based on the scaffold of JX06, the authors identify two novel types of disulfide-based PDK1 inhibitors. The most potent analog, bis(1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3a), effectively inhibits PDK1 both at the mol. (kinact/Ki = 4.17 × 103 M-1s-1) and the cellular level (down to 0.1 μM). In contrast to JX06, 3a is a potent and subtype-selective inhibitor of PDK1 with >40-fold selectivity for PDK2-4. Compd. 3a also significantly alters glucose metabolic pathways in A549 cells by decreasing ECAR and increasing ROS. Moreover, in the xenograft models, 3a shows significant antitumor activity with no neg. effect to the mice wt. Collectively, these data demonstrate that 3a may be an excellent lead compd. for the treatment of cancer as a first-generation subtype-selective and covalent PDK1 inhibitor.
- 243Napolitano, L.; Scalise, M.; Koyioni, M.; Koutentis, P.; Catto, M.; Eberini, I.; Parravicini, C.; Palazzolo, L.; Pisani, L.; Galluccio, M.; Console, L.; Carotti, A.; Indiveri, C. Potent Inhibitors of Human LAT1 (SLC7A5) Transporter Based on Dithiazole and Dithiazine Compounds for Development of Anticancer Drugs. Biochem. Pharmacol. 2017, 143, 39– 52, DOI: 10.1016/j.bcp.2017.07.006[Crossref], [PubMed], [CAS], Google Scholar243https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFOlsL3I&md5=5953170d4a704a9c546775f784ef9fc5Potent inhibitors of human LAT1 (SLC7A5) transporter based on dithiazole and dithiazine compounds for development of anticancer drugsNapolitano, Lara; Scalise, Mariafrancesca; Koyioni, Maria; Koutentis, Panayiotis; Catto, Marco; Eberini, Ivano; Parravicini, Chiara; Palazzolo, Luca; Pisani, Leonardo; Galluccio, Michele; Console, Lara; Carotti, Angelo; Indiveri, CesareBiochemical Pharmacology (Amsterdam, Netherlands) (2017), 143 (), 39-52CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)The LAT1 transporter is acknowledged as a pharmacol. target of tumors since it is strongly overexpressed in many human cancers. The purpose of this work was to find novel compds. exhibiting potent and prolonged inhibition of the transporter. To this aim, compds. based on dithiazole and dithiazine scaffold have been screened in the proteoliposome exptl. model. Inhibition was tested on the antiport catalyzed by hLAT1 as transport of extraliposomal [3H]histidine in exchange with intraliposomal histidine. Out of 59 compds. tested, 8 compds., showing an inhibition higher than 90% at 100 μM concn., were subjected to dose-response anal. Two of them exhibited IC50 lower than 1 μM. Inhibition kinetics, performed on the two best inhibitors, indicated a mixed type of inhibition with respect to the substrate. Furthermore, inhibition of the transporter was still present after removal of the compds. from the reaction mixt., but was reversed on addn. of dithioerythritol, a S-S reducing agent, indicating the formation of disulfide(s) between the compds. and the protein. Mol. docking of the two best inhibitors on the hLAT1 homol. structural model, highlighted interaction with the substrate binding site and formation of a covalent bond with the residue C407. Indeed, the inhibition was impaired in the hLAT1 mutant C407A confirming the involvement of that Cys residue. Treatment of SiHa cells expressing hLAT1 at relatively high level, with the two most potent inhibitors led to cell death which was not obsd. after treatment with a compd. exhibiting very poor inhibitory effect.
- 244Nagy, P. Kinetics and Mechanisms of Thiol–Disulfide Exchange Covering Direct Substitution and Thiol Oxidation-Mediated Pathways. Antioxid. Redox Signaling 2013, 18 (13), 1623– 1641, DOI: 10.1089/ars.2012.4973[Crossref], [PubMed], [CAS], Google Scholar244https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXkvFOjtLo%253D&md5=3e1793928a0494099aabab96413bcf8bKinetics and Mechanisms of Thiol-Disulfide Exchange Covering Direct Substitution and Thiol Oxidation-Mediated PathwaysNagy, PeterAntioxidants & Redox Signaling (2013), 18 (13), 1623-1641CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)A review. Significance: Disulfides are important building blocks in the secondary and tertiary structures of proteins, serving as inter- and intra-subunit cross links. Disulfides are also the major products of thiol oxidn., a process that has primary roles in defense mechanisms against oxidative stress and in redox regulation of cell signaling. Although disulfides are relatively stable, their redn., isomerisation, and interconversion as well as their prodn. reactions are catalyzed by delicate enzyme machineries, providing a dynamic system in biol. Redox homeostasis, a thermodn. parameter that dets. which reactions can occur in cellular compartments, is also balanced by the thiol-disulfide pool. However, it is the kinetic properties of the reactions that best represent cell dynamics, because the partitioning of the possible reactions depends on kinetic parameters. Crit. Issues: This review is focused on the kinetics and mechanisms of thiol-disulfide substitution and redox reactions. It summarizes the challenges and advances that are assocd. with kinetic investigations in small mol. and enzymic systems from a rigorous chem. perspective using biol. examples. The most important parameters that influence reaction rates are discussed in detail. Recent Advances and Future Directions: Kinetic studies of proteins are more challenging than small mols., and quite often investigators are forced to sacrifice the rigor of the exptl. approach to obtain the important kinetic and mechanistic information. However, recent technol. advances allow a more comprehensive anal. of enzymic systems via using the systematic kinetics app. that was developed for small mol. reactions, which is expected to provide further insight into the cell's machinery. Antioxid. Redox Signal. 18, 1623-1641.
- 245Go, Y.-M.; Jones, D. P. Thiol/Disulfide Redox States in Signaling and Sensing. Crit. Rev. Biochem. Mol. Biol. 2013, 48 (2), 173– 181, DOI: 10.3109/10409238.2013.764840[Crossref], [PubMed], [CAS], Google Scholar245https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXkslWntbc%253D&md5=3e9e065adbd2a5b7af0bdd904c5f1719Thiol/disulfide redox states in signaling and sensingGo, Young-Mi; Jones, Dean P.Critical Reviews in Biochemistry and Molecular Biology (2013), 48 (2), 173-181CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare)A review. Rapid advances in redox systems biol. are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol-disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady states displaced from thermodn. equil., that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biol. systems and the knowledge base available to support development of integrated redox systems biol. models to better understand the function and dysfunction of thiol-disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiol. function. Advances in redox proteomics show that, in addn. to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biol. functions. These advances provide a framework for translation of redox systems biol. concepts to practical use in understanding and treating human disease. Biol. responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities.
- 246García-Santamarina, S.; Boronat, S.; Hidalgo, E. Reversible Cysteine Oxidation in Hydrogen Peroxide Sensing and Signal Transduction. Biochemistry 2014, 53 (16), 2560– 2580, DOI: 10.1021/bi401700f[ACS Full Text], [CAS], Google Scholar246https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmt1Gju78%253D&md5=3e5ea91a2214801e4b84066acf71505cReversible Cysteine Oxidation in Hydrogen Peroxide Sensing and Signal TransductionGarcia-Santamarina, Sarela; Boronat, Susanna; Hidalgo, ElenaBiochemistry (2014), 53 (16), 2560-2580CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. Activation of redox cascades through hydrogen peroxide-mediated reversible cysteine oxidn. is a major mechanism for intracellular signaling. Understanding why some cysteine residues are specifically oxidized, in competition with other proximal cysteine residues and in the presence of strong redox buffers, is therefore crucial for understanding redox signaling. In this review, the authors explore the recent advances in thiol-redox chem. linked to signaling. The authors describe the last findings in the field of redox sensors, those that are naturally present in different model organisms as well as those that have been engineered to quantify intracellular hydrogen peroxide concns. Finally, the authors provide a summary of the newest approaches developed to study reversible cysteine oxidn. at the proteomic level.
- 247Gupta, V.; Carroll, K. S. Profiling the Reactivity of Cyclic C-Nucleophiles towards Electrophilic Sulfur in Cysteine Sulfenic Acid. Chem. Sci. 2016, 7 (1), 400– 415, DOI: 10.1039/C5SC02569A[Crossref], [PubMed], [CAS], Google Scholar247https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1WltrnN&md5=249ce8ace72189fc42d75515333afe08Profiling the reactivity of cyclic C-nucleophiles towards electrophilic sulfur in cysteine sulfenic acidGupta, Vinayak; Carroll, Kate S.Chemical Science (2016), 7 (1), 400-415CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Oxidn. of a protein cysteine thiol to sulfenic acid, termed S-sulfenylation, is a reversible post-translational modification that plays a crucial role in regulating protein function and is correlated with disease states. The majority of reaction-based small mol. and immunochem. probes used for detecting sulfenic acids are based on the 5,5-dimethyl-1,3-cyclohexanedione (dimedone) scaffold, which is selective, but suffers from low reactivity. In addn., mechanistic details and features that diminish or enhance nucleophile reactivity remain largely unknown. A significant hurdle to resolving the aforementioned issues has been the chem. unstable nature of small-mol. sulfenic acid models. Herein, we report a facile mass spectrometry-based assay and repurposed dipeptide-based model to screen a library of cyclic C-nucleophiles for reactivity with sulfenic acid under aq. conditions. Obsd. rate consts. for ∼100 cyclic C-nucleophiles were obtained and, from this collection, we have identified novel compds. with more than 200-fold enhanced reactivity, as compared to dimedone. The increase in reactivity and retention of selectivity of these C-nucleophiles were validated in secondary assays, including a protein model for sulfenic acid. Together, this work represents a significant step toward developing new chem. reporters for detecting protein S-sulfenylation with superior kinetic resoln. The enhanced rates and varied compn. of the C-nucleophiles should enable more comprehensive analyses of the sulfenome and serve as the foundation for reversible or irreversible nucleophilic covalent inhibitors that target oxidized cysteine residues in therapeutically important proteins.
- 248Truong, T. H.; Carroll, K. S. Redox Regulation of Epidermal Growth Factor Receptor Signaling through Cysteine Oxidation. Biochemistry 2012, 51 (50), 9954– 9965, DOI: 10.1021/bi301441e[ACS Full Text], [CAS], Google Scholar248https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhslelsr7O&md5=80ce6e8d25c413b3e4040fd391f87555Redox regulation of epidermal growth factor receptor signaling through cysteine oxidationTruong, Thu H.; Carroll, Kate S.Biochemistry (2012), 51 (50), 9954-9965CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. Epidermal growth factor receptor (EGFR) exemplifies the family of receptor tyrosine kinases that mediate numerous cellular processes, including growth, proliferation, and differentiation. Moreover, gene amplification and EGFR mutations have been identified in a no. of human malignancies, making this receptor an important target for the development of anticancer drugs. In addn. to ligand-dependent activation and concomitant tyrosine phosphorylation, EGFR stimulation results in the localized generation of H2O2 by NADPH-dependent oxidases. In turn, H2O2 functions as a secondary messenger to regulate intracellular signaling cascades, largely through the modification of specific cysteine residues within redox-sensitive protein targets, including Cys797 in the EGFR active site. In this review, we highlight recent advances in our understanding of the mechanisms that underlie redox regulation of EGFR signaling and how these discoveries may form the basis for the development of new therapeutic strategies for targeting this and other H2O2-modulated pathways.
- 249Garcia, F. J.; Carroll, K. S. Redox-Based Probes as Tools to Monitor Oxidized Protein Tyrosine Phosphatases in Living Cells. Eur. J. Med. Chem. 2014, 88, 28– 33, DOI: 10.1016/j.ejmech.2014.06.040[Crossref], [PubMed], [CAS], Google Scholar249https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVOju7vP&md5=221c3c815cb7fd727ce86ea5b88a0c06Redox-based probes as tools to monitor oxidized protein tyrosine phosphatases in living cellsGarcia, Francisco J.; Carroll, Kate S.European Journal of Medicinal Chemistry (2014), 88 (), 28-33CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Reversible oxidn. of protein tyrosine phosphatases (PTPs) has emerged as an important regulatory mechanism whereby reactive oxygen species (ROS) inactivates the PTP and promotes phosphorylation and induction of the signaling cascade. The lack of sensitive and robust methods to directly detect oxidized PTPs has made it difficult to understand the effects that PTP oxidative inactivation play in redox signaling. We report the use of redox-based probes to directly detect oxidized PTPs in a cellular context, which highlights the importance of direct approaches to assist in the study of physiol. and pathophysiol. PTP activity in redox regulation. We also demonstrate, as a proof-of-concept, that these redox-based probes serve as prototypes for the design and development of a new class of inhibitors for phosphatases. We envision a nucleophile reacting with the oxidized inactive catalytic cysteine to generate an irreversible thioether adduct which prevents the phosphatase from being reactivated and ultimately fortifies the signaling cascade. Our results reveal the potential of translation of our redox-based probes, which are used to understand redox cell circuitry and disease biol., to small-mol. nucleophile-based inhibitors, which may treat diseases assocd. with redox stress. This may have implications in the treatment of type 2 diabetes and cancer.
- 250Alcock, L. J.; Farrell, K. D.; Akol, M. T.; Jones, G. H.; Tierney, M. M.; Kramer, H. B.; Pukala, T. L.; Bernardes, G. J. L.; Perkins, M. V.; Chalker, J. M. Norbornene Probes for the Study of Cysteine Oxidation. Tetrahedron 2018, 74 (12), 1220– 1228, DOI: 10.1016/j.tet.2017.11.011[Crossref], [CAS], Google Scholar250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVWqurbE&md5=bcf72e863b0040d346a907351c72409fNorbornene probes for the study of cysteine oxidationAlcock, Lisa J.; Farrell, Kyle D.; Akol, Mawey T.; Jones, Gregory H.; Tierney, Matthew M.; Kramer, Holger B.; Pukala, Tara L.; Bernardes, Goncalo J. L.; Perkins, Michael V.; Chalker, Justin M.Tetrahedron (2018), 74 (12), 1220-1228CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)Cysteine residues on proteins can react with cellular oxidants such as hydrogen peroxide. While this process is important for scavenging excess reactive oxygen species, the products of this oxidn. may also mediate cell signaling. To understand the role of cysteine oxidn. in biol., selective probes are required to detect and quantify its occurrence. Cysteine oxidn. products such as sulfenic acids are sometimes unstable and therefore short-lived. If such cysteine derivs. are to be analyzed, rapid reaction with the probe is required. Here the authors introduce norbornene derivs. as probes for cysteine oxidn., and demonstrate their ability to trap sulfenic acids. The synthesis of norbornene derivs. contg. alkyne or biotin affinity tags are also reported to facilitate the use of these probes in chem. biol. and proteomics.
- 251Poole, T. H.; Reisz, J. A.; Zhao, W.; Poole, L. B.; Furdui, C. M.; King, S. B. Strained Cycloalkynes as New Protein Sulfenic Acid Traps. J. Am. Chem. Soc. 2014, 136 (17), 6167– 6170, DOI: 10.1021/ja500364r[ACS Full Text], [CAS], Google Scholar251https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtVegtL0%253D&md5=af7b13df6e6bd4101306df5be38bd88aStrained Cycloalkynes as New Protein Sulfenic Acid TrapsPoole, Thomas H.; Reisz, Julie A.; Zhao, Weiling; Poole, Leslie B.; Furdui, Cristina M.; King, S. BruceJournal of the American Chemical Society (2014), 136 (17), 6167-6170CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein sulfenic acids are formed by the reaction of biol. relevant reactive oxygen species with protein thiols. Sulfenic acid formation modulates the function of enzymes and transcription factors either directly or through the subsequent formation of protein disulfide bonds. Identifying the site, timing, and conditions of protein sulfenic acid formation remains crucial to understanding cellular redox regulation. Current methods for trapping and analyzing sulfenic acids involve the use of dimedone and other nucleophilic 1,3-dicarbonyl probes that form covalent adducts with cysteine-derived protein sulfenic acids. As a mechanistic alternative, the present study describes highly strained bicyclo[6.1.0]nonyne (BCN) derivs. as concerted traps of sulfenic acids. These strained cycloalkynes react efficiently with sulfenic acids in proteins and small mols. yielding stable alkenyl sulfoxide products at rates >100x greater than 1,3-dicarbonyl reagents enabling kinetic competition with physiol. sulfur chem. Similar to the 1,3-dicarbonyl reagents, the BCN compds. distinguish the sulfenic acid oxoform from the thiol, disulfide, sulfinic acid, and S-nitrosated forms of cysteine while displaying an acceptable cell toxicity profile. The enhanced rates demonstrated by these strained alkynes identify them as new bioorthogonal probes that should facilitate the discovery of previously unknown sulfenic acid sites and their parent proteins.
- 252Gupta, V.; Carroll, K. S. Rational Design of Reversible and Irreversible Cysteine Sulfenic Acid-Targeted Linear C-Nucleophiles. Chem. Commun. 2016, 52 (16), 3414– 3417, DOI: 10.1039/C6CC00228E[Crossref], [PubMed], [CAS], Google Scholar252https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtl2ns7g%253D&md5=4279f7d9100cadfe4747bfebf97c2845Rational design of reversible and irreversible cysteine sulfenic acid-targeted linear C-nucleophilesGupta, Vinayak; Carroll, Kate S.Chemical Communications (Cambridge, United Kingdom) (2016), 52 (16), 3414-3417CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Concerns about off-target effects has motivated the development of reversible covalent inhibition strategies for targeting cysteine. However, such strategies have not been reported for the unique cysteine oxoform, sulfenic acid. Herein, we have designed and identified linear C-nucleophiles that react selectively with cysteine sulfenic acid. The resulting thioether adducts exhibit reversibility ranging from minutes to days under reducing conditions, showing the feasibility of tuning C-nucleophile reactivity across a wide range of time scales.
- 253Forman, H. J.; Davies, M. J.; Krämer, A. C.; Miotto, G.; Zaccarin, M.; Zhang, H.; Ursini, F. Protein Cysteine Oxidation in Redox Signaling: Caveats on Sulfenic Acid Detection and Quantification. Arch. Biochem. Biophys. 2017, 617, 26– 37, DOI: 10.1016/j.abb.2016.09.013[Crossref], [PubMed], [CAS], Google Scholar253https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1WntrjF&md5=d37c64fc4beecbb6d8349255064f6236Protein cysteine oxidation in redox signaling: Caveats on sulfenic acid detection and quantificationForman, Henry Jay; Davies, Michael J.; Kramer, Anna C.; Miotto, Giovanni; Zaccarin, Mattia; Zhang, Hongqiao; Ursini, FulvioArchives of Biochemistry and Biophysics (2017), 617 (), 26-37CODEN: ABBIA4; ISSN:0003-9861. (Elsevier B.V.)Oxidn. of crit. signaling protein cysteines regulated by H2O2 has been considered to involve sulfenic acid (RSOH) formation. RSOH may subsequently form either a sulfenyl amide (RSNHR') with a neighboring amide, or a mixed disulfide (RSSR') with another protein cysteine or glutathione. Previous studies have claimed that RSOH can be detected as an adduct (e.g., with 5,5-dimethylcyclohexane-1,3-dione; dimedone). Here, kinetic data are discussed which indicate that few proteins can form RSOH under physiol. signaling conditions. We also present exptl. evidence that indicates that (1) dimedone reacts rapidly with sulfenyl amides, and more rapidly than with sulfenic acids, and (2) that disulfides can react reversibly with amides to form sulfenyl amides. As some proteins are more stable as the sulfenyl amide than as a glutathionylated species, the former may account for some of the species previously identified as the "sulfenome" - the cellular complement of reversibly-oxidized thiol proteins generated via sulfenic acids.
- 254Gupta, V.; Yang, J.; Liebler, D. C.; Carroll, K. S. Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles. J. Am. Chem. Soc. 2017, 139 (15), 5588– 5595, DOI: 10.1021/jacs.7b01791[ACS Full Text], [CAS], Google Scholar254https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXltlCmsbY%253D&md5=ac0b22e7270f452df5851033ae73e126Diverse Redoxome Reactivity Profiles of Carbon NucleophilesGupta, Vinayak; Yang, Jing; Liebler, Daniel C.; Carroll, Kate S.Journal of the American Chemical Society (2017), 139 (15), 5588-5595CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this process is the identification of signaling pathways (or receptors) specific to (or overexpressed in) disease cells. In this context, fragment-based ligand discovery (FBLD) has significantly expanded our view of the ligandable proteome and affords tool compds. for biol. inquiry. To date, such covalent ligand discovery has almost exclusively employed cysteine-reactive small-mol. fragments. However, functional cysteine residues in proteins are often redox-sensitive and can undergo oxidn. in cells. Such reactions are particularly relevant in diseases, like cancer, which are linked to excessive prodn. of reactive oxygen species. Once oxidized, the sulfur atom of cysteine is much less reactive toward electrophilic groups used in the traditional FBLD paradigm. To address this limitation, we recently developed a novel library of diverse carbon-based nucleophile fragments that react selectively with cysteine sulfenic acid formed in proteins via oxidn. or hydrolysis reactions. Here, we report anal. of sulfenic acid-reactive C-nucleophile fragments screened against a colon cancer cell proteome. Covalent ligands were identified for >1280 S-sulfenylated cysteines present in "druggable" proteins and orphan targets, revealing disparate reactivity profiles and target preferences. Among the unique ligand-protein interactions identified was that of a pyrrolidinedione nucleophile that reacted preferentially with protein tyrosine phosphatases. Fragment-based covalent ligand discovery with C-nucleophiles affords an expansive snapshot of the ligandable "redoxome" with significant implications for covalent inhibitor pharmacol. and also affords new chem. tools to investigate redox-regulation of protein function.
- 255Holliday, G. L.; Mitchell, J. B. O.; Thornton, J. M. Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis. J. Mol. Biol. 2009, 390 (3), 560– 577, DOI: 10.1016/j.jmb.2009.05.015[Crossref], [PubMed], [CAS], Google Scholar255https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnsl2itbc%253D&md5=15be47b78d45b7d3c8f393e3bd20fc20Understanding the functional roles of amino acid residues in enzyme catalysisHolliday, Gemma L.; Mitchell, John B. O.; Thornton, Janet M.Journal of Molecular Biology (2009), 390 (3), 560-577CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The MACiE database contains 223 distinct step-wise enzyme reaction mechanisms and holds representatives from each EC sub-subclass where there is a crystal structure and sufficient evidence in the literature to support a mechanism. Each catalytic step of every reaction sequence in MACiE is fully annotated so that it includes the function of the catalytic residues involved in the reaction and the mechanism by which substrates are transformed into products. Using MACiE as a knowledge base, the authors have seen that the top 10 most catalytic residues are His, Asp, Glu, Lys, Cys, Arg, Ser, Thr, Tyr, and Trp. Of these, only 7 (Cys, His, Asp, Lys, Ser, Thr, and Tyr) dominate catalysis and provide essentially 5 functional roles that are essential. Stabilization is the most common and essential role for all classes of enzyme, followed by general acid/base (proton acceptor and proton donor) functionality, with nucleophilic addn. following closely behind (nucleophile and nucleofuge). The authors investigated the occurrence of these residues in MACiE and the Catalytic Site Atlas and found that, as expected, certain residue types were assocd. with each functional role, with some residue types able to perform diverse roles. In addn., it was seen that different EC classes of enzyme had a tendency to employ different residues for catalysis. Further, the authors showed that while the differences between EC classes in catalytic residue compn. were not immediately obvious from the general classes of Ingold mechanisms, there was some weak correlation between the mechanisms involved in a given EC class and the functions that the catalytic amino acid residues were performing. The anal. presented here provides a valuable insight into the functional roles of catalytic amino acid residues, which may have applications in many aspects of enzymol., from the design of novel enzymes to the prediction and validation of enzyme reaction mechanisms.
- 256Platzer, G.; Okon, M.; McIntosh, L. P. PH-Dependent Random Coil 1H, 13C, and 15N Chemical Shifts of the Ionizable Amino Acids: A Guide for Protein PKa Measurements. J. Biomol. NMR 2014, 60 (2–3), 109– 129, DOI: 10.1007/s10858-014-9862-y[Crossref], [PubMed], [CAS], Google Scholar256https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFOqtbjM&md5=196361f2a4e7a2ba970a50844c578cdbpH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pKa measurementsPlatzer, Gerald; Okon, Mark; McIntosh, Lawrence P.Journal of Biomolecular NMR (2014), 60 (2-3), 109-129CODEN: JBNME9; ISSN:0925-2738. (Springer)The pKa values and charge states of ionizable residues in polypeptides and proteins are frequently detd. via NMR-monitored pH titrns. To aid the interpretation of the resulting titrn. data, we have measured the pH-dependent chem. shifts of nearly all the 1H, 13C, and 15N nuclei in the seven common ionizable amino acids (X = Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, resp. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor ref. chem. shifts to help guide protein pKa measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chem. shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain 15N nuclei of His, Lys, and Arg in their pos.-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary exptl. evidence for defining the ionization states of protein residues.
- 257Isom, D. G.; Castañeda, C. A.; Cannon, B. R.; Garcia-Moreno, B. E. Large Shifts in PKa Values of Lysine Residues Buried inside a Protein. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (13), 5260– 5265, DOI: 10.1073/pnas.1010750108[Crossref], [PubMed], [CAS], Google Scholar257https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkslOmt7c%253D&md5=aefb9e5d2cb9362ff63688fab588f7b0Large shifts in pKa values of lysine residues buried inside a proteinIsom, Daniel G.; Castaneda, Carlos A.; Cannon, Brian R.; Garcia-Moreno, Bertrand E.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (13), 5260-5265, S5260/1-S5260/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine mol. determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pKa values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pKa values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biol. H+ transport. The pKa values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pKa values varied significantly from location to location inside the protein. These data will enable an unprecedented examn. of the positional dependence of the dielec. response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function.
- 258Hacker, S. M.; Backus, K. M.; Lazear, M. R.; Forli, S.; Correia, B. E.; Cravatt, B. F. Global Profiling of Lysine Reactivity and Ligandability in the Human Proteome. Nat. Chem. 2017, 9 (12), 1181– 1190, DOI: 10.1038/nchem.2826[Crossref], [PubMed], [CAS], Google Scholar258https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1GrsrrN&md5=1dac8f079c1caffc8ae584291480fc61Global profiling of lysine reactivity and ligandability in the human proteomeHacker, Stephan M.; Backus, Keriann M.; Lazear, Michael R.; Forli, Stefano; Correia, Bruno E.; Cravatt, Benjamin F.Nature Chemistry (2017), 9 (12), 1181-1190CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, the authors report a chem. proteomic platform for the global and quant. anal. of lysine residues in native biol. systems. The authors have quantified, in total, >9000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small mols. The authors also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.
- 259Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Structural Determinants of Phosphoinositide 3-Kinase Inhibition by Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine. Mol. Cell 2000, 6 (4), 909– 919, DOI: 10.1016/S1097-2765(05)00089-4[Crossref], [PubMed], [CAS], Google Scholar259https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnvVegurY%253D&md5=5dd92f9b48c437e43c0023fdff10c1acStructural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporineWalker, Edward H.; Pacold, Michael E.; Perisic, Olga; Stephens, Len; Hawkins, Philip T.; Wymann, Matthias P.; Williams, Roger L.Molecular Cell (2000), 6 (4), 909-919CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)The specific phosphoinositide 3-kinase (P13K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallog. structures of P13K bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compds. fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compd. on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind P13K in remarkably different orientations that are related to each other by 1800 rotations. Staurosporine/P13K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific P13K inhibitors with therapeutic potential.
- 260Wymann, M. P.; Bulgarelli-Leva, G.; Zvelebil, M. J.; Pirola, L.; Vanhaesebroeck, B.; Waterfield, M. D.; Panayotou, G. Wortmannin Inactivates Phosphoinositide 3-Kinase by Covalent Modification of Lys-802, a Residue Involved in the Phosphate Transfer Reaction. Mol. Cell. Biol. 1996, 16 (4), 1722– 1733, DOI: 10.1128/MCB.16.4.1722[Crossref], [PubMed], [CAS], Google Scholar260https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XhvVags7s%253D&md5=54ebe365b4ad8260555c8b962e03d28bWortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reactionWymann, Matthias P.; Bulgarelli-Leva, Ginette; Zvelebil, Marketa J.; Pirola, Luciano; Vanhaesebroeck, Bart; Waterfield, Michael D.; Panayotou, GeorgeMolecular and Cellular Biology (1996), 16 (4), 1722-33CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Wortmannin at nanomolar concns. is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concns., wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA dependent protein kinase (DNA-PKcs). The authors report here the identification of the site of interaction of wortmannin in the catalytic subunit of PI3-kinase, p110α. At physiol. pH (6.5 to 8) wortmannin reacted specifically with p110α. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances contg. nucleophilic amino acid side chain functions had no effect at the same concns. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant P110α were mapped with anti-wortmannin and anti-p110α peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-59-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-t9-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. The results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiol. processes.
- 261Pettinger, J.; Le Bihan, Y.-V.; Widya, M.; van Montfort, R. L. M.; Jones, K.; Cheeseman, M. D. An Irreversible Inhibitor of HSP72 That Unexpectedly Targets Lysine-56. Angew. Chem., Int. Ed. 2017, 56 (13), 3536– 3540, DOI: 10.1002/anie.201611907[Crossref], [CAS], Google Scholar261https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjtVeru7o%253D&md5=8b2cbd17140e5f0c6430d0164e03280aAn Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine-56Pettinger, Jonathan; Le Bihan, Yann-Vai; Widya, Marcella; van Montfort, Rob L. M.; Jones, Keith; Cheeseman, Matthew D.Angewandte Chemie, International Edition (2017), 56 (13), 3536-3540CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The stress-inducible mol. chaperone, HSP72, is an important therapeutic target in oncol., but inhibiting this protein with small mols. has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine-targeted irreversible inhibitor. Using rational design, we adapted a validated 8-N-benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine-56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chem. probes and drugs.
- 262Altmeyer, M.; Amtmann, E.; Heyl, C.; Marschner, A.; Scheidig, A. J.; Klein, C. D. Beta-Aminoketones as Prodrugs for Selective Irreversible Inhibitors of Type-1 Methionine Aminopeptidases. Bioorg. Med. Chem. Lett. 2014, 24 (22), 5310– 5314, DOI: 10.1016/j.bmcl.2014.09.047[Crossref], [PubMed], [CAS], Google Scholar262https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1eitbjK&md5=e7b2d0e83fb348fdbbb59f622d5fa918Beta-aminoketones as prodrugs for selective irreversible inhibitors of type-1 methionine aminopeptidasesAltmeyer, Markus; Amtmann, Eberhard; Heyl, Carina; Marschner, Aline; Scheidig, Axel J.; Klein, Christian D.Bioorganic & Medicinal Chemistry Letters (2014), 24 (22), 5310-5314CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)We identified and characterized β-aminoketones as prodrugs for irreversible MetAP inhibitors that are selective for the MetAP-1 subtype. β-Aminoketones with certain structural features form α,β-unsatd. ketones under physiol. conditions, which bind covalently and selectively to cysteines in the S1 pocket of MetAP-1. The binding mode was confirmed by X-ray crystallog. and assays with the MetAPs from Escherichia coli, Staphylococcus aureus and both human isoforms. The initially identified tetralone derivs. showed complete selectivity for E. coli MetAP vs. human MetAP-1 and MetAP-2. Rational design of indanone analogs yielded compds. with selectivity for the human type-1 vs. the human type-2 MetAP.
- 263Dahal, U. P.; Gilbert, A. M.; Obach, R. S.; Flanagan, M. E.; Chen, J. M.; Garcia-Irizarry, C.; Starr, J. T.; Schuff, B.; Uccello, D. P.; Young, J. A. Intrinsic Reactivity Profile of Electrophilic Moieties to Guide Covalent Drug Design: N-α-Acetyl-L-Lysine as an Amine Nucleophile. MedChemComm 2016, 7 (5), 864– 872, DOI: 10.1039/C6MD00017G[Crossref], [CAS], Google Scholar263https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XisFyjtrk%253D&md5=08e681ea8dfb353f89d8cdedba685694Intrinsic reactivity profile of electrophilic moieties to guide covalent drug design: N-α-acetyl-L-lysine as an amine nucleophileDahal, Upendra P.; Gilbert, Adam M.; Obach, R. Scott; Flanagan, Mark E.; Chen, Jinshan M.; Garcia-Irizarry, Carmen; Starr, Jeremy T.; Schuff, Brandon; Uccello, Daniel P.; Young, Jennifer A.MedChemComm (2016), 7 (5), 864-872CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)Covalent drugs contain a reactive electrophilic moiety or covalent reactive group (CRG), which forms an irreversible bond between the drug and a biol. target. Consequently, the intrinsic reactivity of the CRG is an important consideration in the design of irreversible inhibitors. Although reactivity assessments of CRGs with sulfur nucleophiles, such as glutathione and cysteine have been reported, reactivity of these moieties with amine-contg. nucleophiles is not well described. In this study, intrinsic reactivities were detd. for a series of electrophiles (acrylamides, nitriles, cyanamides, sulfones, and sulfonamides) using N-α-acetyl-L-lysine as a model amine-based nucleophile and compared with results using glutathione (GSH). Since the ε-amine of N-α-acetyl-L-lysine is protonated at neutral pH, reactions were carried out at pH 10.2. In addn. to reporting rate data for reactions of CRGs with N-α-acetyl-L-lysine, elements of selectivity relative to thiol-contg. nucleophiles are also be discussed.
- 264Anscombe, E.; Meschini, E.; Mora-Vidal, R.; Martin, M. P.; Staunton, D.; Geitmann, M.; Danielson, U. H.; Stanley, W. A.; Wang, L. Z.; Reuillon, T.; Golding, B. T.; Cano, C.; Newell, D. R.; Noble, M. E. M.; Wedge, S. R.; Endicott, J. A.; Griffin, R. J. Identification and Characterization of an Irreversible Inhibitor of CDK2. Chem. Biol. 2015, 22 (9), 1159– 1164, DOI: 10.1016/j.chembiol.2015.07.018[Crossref], [PubMed], [CAS], Google Scholar264https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVajsL7M&md5=e6a29b14eb31abd659a48511bb11ed98Identification and Characterization of an Irreversible Inhibitor of CDK2Anscombe, Elizabeth; Meschini, Elisa; Mora-Vidal, Regina; Martin, Mathew P.; Staunton, David; Geitmann, Matthis; Danielson, U. Helena; Stanley, Will A.; Wang, Lan Z.; Reuillon, Tristan; Golding, Bernard T.; Cano, Celine; Newell, David R.; Noble, Martin E. M.; Wedge, Stephen R.; Endicott, Jane A.; Griffin, Roger J.Chemistry & Biology (Oxford, United Kingdom) (2015), 22 (9), 1159-1164CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclohexylmethoxy)-9H-purin-2-yl)amino)benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl)phenyl)-9H-purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compds.
- 265Narayanan, A.; Jones, L. H. Sulfonyl Fluorides as Privileged Warheads in Chemical Biology. Chem. Sci. 2015, 6 (5), 2650– 2659, DOI: 10.1039/C5SC00408J[Crossref], [PubMed], [CAS], Google Scholar265https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksVamurc%253D&md5=0009feb4068e912b4784fb8411cdb702Sulfonyl fluorides as privileged warheads in chemical biologyNarayanan, Arjun; Jones, Lyn H.Chemical Science (2015), 6 (5), 2650-2659CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Sulfonyl fluoride electrophiles have found significant utility as reactive probes in chem. biol. and mol. pharmacol. As warheads they possess the right balance of biocompatibility (including aq. stability) and protein reactivity. Their functionality is privileged in this regard as they are known to modify not only reactive serines (resulting in their common use as protease inhibitors), but also context-specific threonine, lysine, tyrosine, cysteine and histidine residues. This review describes the application of sulfonyl fluoride probes across various areas of research and explores new approaches that could further enhance the chem. biol. toolkit. We believe that sulfonyl fluoride probes will find greater utility in areas such as covalent enzyme inhibition, target identification and validation, and the mapping of enzyme binding sites, substrates and protein-protein interactions.
- 266Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B. Sulfur(VI) Fluoride Exchange (SuFEx): Another Good Reaction for Click Chemistry. Angew. Chem., Int. Ed. 2014, 53 (36), 9430– 9448, DOI: 10.1002/anie.201309399[Crossref], [CAS], Google Scholar266https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtleisbzJ&md5=78e251c607d42f2506a9420f2f2f9835Sulfur(VI) Fluoride Exchange (SuFEx): Another Good Reaction for Click ChemistryDong, Jiajia; Krasnova, Larissa; Finn, M. G.; Sharpless, K. BarryAngewandte Chemie, International Edition (2014), 53 (36), 9430-9448CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Aryl sulfonyl chlorides (e.g. Ts-Cl) are beloved of org. chemists as the most commonly used SVI electrophiles, and the parent sulfuryl chloride, O2SVICl2, also was relied on to create sulfates and sulfamides. However, the desired halide substitution event is often defeated by destruction of the sulfur electrophile because the SVI-Cl bond is exceedingly sensitive to reductive collapse yielding SIV species and Cl-. Fortunately, the use of sulfur(VI) fluorides (e.g., R-SO2-F and SO2F2) leaves only the substitution pathway open. As with most of click chem., many essential features of sulfur(VI) fluoride reactivity were discovered long ago in Germany. Surprisingly, this extraordinary work faded from view rather abruptly in the mid-20th century. Here the authors seek to revive it, along with John Hyatt's unnoticed 1979 full paper exposition on CH2=CH-SO2-F, the most perfect Michael acceptor ever found. To this history the authors add several new observations, including that the otherwise very stable gas SO2F2 has excellent reactivity under the right circumstances. Also proton or silicon centers can activate the exchange of S-F bonds for S-O bonds to make functional products, and the sulfate connector is surprisingly stable toward hydrolysis. Applications of this controllable ligation chem. to small mols., polymers, and biomols. are discussed.
- 267Baker, B. R. Irreversible Enzyme Inhibitors. CXLIX. Tissue-Specific Irreversible Inhibitors of Dihydrofolic Reductase. Acc. Chem. Res. 1969, 2 (5), 129– 136, DOI: 10.1021/ar50017a001[ACS Full Text], [CAS], Google Scholar267https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF1MXksVKjsL4%253D&md5=bc7effcdf83f4fae3ec4a90f971ea4f0Irreversible enzyme inhibitors. CXLIX. Tissue-specific irreversible inhibitors of dihydrofolic reductaseBaker, Bernard RandallAccounts of Chemical Research (1969), 2 (5), 129-36CODEN: ACHRE4; ISSN:0001-4842.Inhibitors that partly complex in the active site and partly complex in the hydrophobic bonding region can be constructed to show huge differences in binding between mammalian enzymes on one hand and protozoal enzymes on the other. This is of chemotherapeutic utility. 17 references.
- 268Chen, W.; Dong, J.; Plate, L.; Mortenson, D. E.; Brighty, G. J.; Li, S.; Liu, Y.; Galmozzi, A.; Lee, P. S.; Hulce, J. J.; Cravatt, B. F.; Saez, E.; Powers, E. T.; Wilson, I. A.; Sharpless, K. B.; Kelly, J. W. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue. J. Am. Chem. Soc. 2016, 138 (23), 7353– 7364, DOI: 10.1021/jacs.6b02960[ACS Full Text], [CAS], Google Scholar268https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XotVylu7Y%253D&md5=d946cba154b07a712ceae9e8136f8480Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr ResidueChen, Wentao; Dong, Jiajia; Plate, Lars; Mortenson, David E.; Brighty, Gabriel J.; Li, Suhua; Liu, Yu; Galmozzi, Andrea; Lee, Peter S.; Hulce, Jonathan J.; Cravatt, Benjamin F.; Saez, Enrique; Powers, Evan T.; Wilson, Ian A.; Sharpless, K. Barry; Kelly, Jeffery W.Journal of the American Chemical Society (2016), 138 (23), 7353-7364CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-mol. ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis expts., provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function.
- 269Mortenson, D. E.; Brighty, G. J.; Plate, L.; Bare, G.; Chen, W.; Li, S.; Wang, H.; Cravatt, B. F.; Forli, S.; Powers, E. T.; Sharpless, K. B.; Wilson, I. A.; Kelly, J. W. “Inverse Drug Discovery” Strategy To Identify Proteins That Are Targeted by Latent Electrophiles As Exemplified by Aryl Fluorosulfates. J. Am. Chem. Soc. 2018, 140 (1), 200– 210, DOI: 10.1021/jacs.7b08366[ACS Full Text], [CAS], Google Scholar269https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitVShtLbF&md5=cfeb5f1ae67fe89340fffaa4cf5e46a8"Inverse Drug Discovery" Strategy To Identify Proteins That Are Targeted by Latent Electrophiles As Exemplified by Aryl FluorosulfatesMortenson, David E.; Brighty, Gabriel J.; Plate, Lars; Bare, Grant; Chen, Wentao; Li, Suhua; Wang, Hua; Cravatt, Benjamin F.; Forli, Stefano; Powers, Evan T.; Sharpless, K. Barry; Wilson, Ian A.; Kelly, Jeffery W.Journal of the American Chemical Society (2018), 140 (1), 200-210CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Drug candidates are generally discovered using biochem. screens employing an isolated target protein or by utilizing cell-based phenotypic assays. Both noncovalent and covalent hits emerge from such endeavors. Herein, we exemplify an "Inverse Drug Discovery" strategy in which org. compds. of intermediate complexity harboring weak, but activatable, electrophiles are matched with the protein(s) they react with in cells or cell lysate. An alkyne substructure in each candidate small mol. enables affinity chromatog.-mass spectrometry, which produces a list of proteins that each distinct compd. reacts with. A notable feature of this approach is that it is agnostic with respect to the cellular proteins targeted. To illustrate this strategy, we employed aryl fluorosulfates, an underexplored class of sulfur(VI) halides, that are generally unreactive unless activated by protein binding. Reversible aryl fluorosulfate binding, correct juxtaposition of protein side chain functional groups, and transition-state stabilization of the S(VI) exchange reaction all seem to be crit. for conjugate formation. The aryl fluorosulfates studied thus far exhibit chemoselective reactivity toward Lys and, particularly, Tyr side chains, and can be used to target nonenzymes (e.g., a hormone carrier or a small-mol. carrier protein) as well as enzymes. The "Inverse Drug Discovery" strategy should be particularly attractive as a means to explore latent electrophiles not typically used in medicinal chem. efforts, until one reacts with a protein target of exceptional interest. Structure-activity data can then be used to enhance the selectivity of conjugate formation or the covalent probe can be used as a competitor to develop noncovalent drug candidates. Here we use the "Inverse Drug Discovery" platform to identify and validate covalent ligands for 11 different human proteins. In the case of one of these proteins, we have identified and validated a small-mol. probe for the first time.
- 270James, G. T. Inactivation of the Protease Inhibitor Phenylmethylsulfonyl Fluoride in Buffers. Anal. Biochem. 1978, 86 (2), 574– 579, DOI: 10.1016/0003-2697(78)90784-4[Crossref], [PubMed], [CAS], Google Scholar270https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXktF2jtr8%253D&md5=71a4087d48cfdee57eb4d70c9e8b7d2fInactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffersJames, Gordon T.Analytical Biochemistry (1978), 86 (2), 574-9CODEN: ANBCA2; ISSN:0003-2697.Aq. prepns. of phenylmethylsulfonyl fluoride (PMSF) are inactive toward proteases unless promptly brought into contact with protease. Inactivation of PMSF increased with increased pH and temp. Halflives of the inhibitor at 25° were ∼110, 55, and 35 min at pH 7.0, 7.5, and 8.0, resp. At pH 8,100 μM PMSF was almost completely inactivated within 1 h at 25° or within 22 h at 4°. Stock solns. of PMSF in 100% isopropanol were stable at 25° for many months. Reactivation of PMSF-inhibited chymotrypsin did not occur within 1 wk at 25° at pH 7.0.
- 271Lively, M. O.; Powers, J. C. Specificity and Reactivity of Human Granulocyte Elastase and Cathepsin G, Porcine Pancreatic Elastase, Bovine Chymotrypsin and Trypsin toward Inhibition with Sulfonyl Flourides. Biochim. Biophys. Acta BBA - Enzymol. 1978, 525 (1), 171– 179, DOI: 10.1016/0005-2744(78)90211-5
- 272Genov, N. C.; Shopova, M.; Boteva, R.; Ricchelli, F.; Jori, G. Intramolecular Distances between Tryptophan Residues and the Active-Site Serine Residue in Alkaline Bacterial Proteinases as Measured by Fluorescence Energy-Transfer Studies. Biochem. J. 1983, 215 (2), 413– 416, DOI: 10.1042/bj2150413[Crossref], [PubMed], [CAS], Google Scholar272https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXmtVykurk%253D&md5=3db650c76b172dfe1e3f21c2f125361dIntramolecular distances between tryptophan residues and the active-site serine residue in alkaline bacterial proteinases as measured by fluorescence energy-transfer studiesGenov, N.; Shopova, M.; Boteva, R.; Ricchelli, F.; Jori, G.Biochemical Journal (1983), 215 (2), 413-16CODEN: BIJOAK; ISSN:0264-6021.Singlet-singlet energy transfer from the tryptophan residues to an active-site-serine-bound 5-dimethylaminonaphthalene-1-sulfonyl group was investigated in 4 subtilisins. The transfer distance for subtilisin Novo and mesentericopeptidase are 1.93 and 1.81 nm, resp. The positions of the indole groups in the 3-dimensional structures of 2 pairs of proteinases, namely subtilisin Novo and mesentericopeptidase on the one hand and subtilisins Carlsberg and DY on the other, are essentially identical.
- 273Esch, F. S.; Allison, W. S. Identification of a Tyrosine Residue at a Nucleotide Binding Site in the Beta Subunit of the Mitochondrial ATPase with P-Fluorosulfonyl[14C]-Benzoyl-5′-Adenosine. J. Biol. Chem. 1978, 253, 6100– 6106[PubMed], [CAS], Google Scholar273https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXlslGlurc%253D&md5=a04ea874870c890bca5148857ebe364eIdentification of a tyrosine residue at a nucleotide binding site in the β subunit of the mitochondrial ATPase with p-fluorosulfonyl[14C]-benzoyl-5'-adenosineEsch, Fred S.; Allison, William S.Journal of Biological Chemistry (1978), 253 (17), 6100-6CODEN: JBCHA3; ISSN:0021-9258.Bovine heart mitochondrial F1-ATPase is irreversibly inactivated by the adenine nucleotide analog, p-fluorosulfonylbenzoyl-5'-adenosine (I). This inactivation is partly prevented by the presence of bound adenine nucleotides. Inactivations of the ATPase with I-14C were most efficiently accomplished with the nucleotide-free enzyme at pH 7.0, in a buffer contg. 20% glycerol. Under these conditions, 4.2 g-atoms of 14C are incorporated/350,000 g enzyme when the ATPase is inactivated by 90% by its reaction with 2 mM I-14C. Isolation of the component polypeptide chains of the labeled ATPase showed that all of the radioactivity was assocd. with the 2 largest subunits. The isolated α subunit contained 0.45 g-atom 14C/mol and the isolated β subunit contained 0.88 g atom 14C/mol. Hence, the inactivation can be correlated with the incorporation of 14C into the β subunit. This suggests that the hydrolytic site of the enzyme resides on this subunit. The majority of the radioactivity in a tryptic digest of labeled β subunit is contained in a tryptic peptide that has the following amino acid sequence: Ile-Met-Asp-Pro-Asn-Ile-Val-Gly-Ser-Glu-His-*Tyr-Asp-Val-Ala-Arg, where *Tyr is the radioactive deriv. of the tyrosine residue that was sulfonylated during the inactivation.
- 274Jörg, M.; Glukhova, A.; Abdul-Ridha, A.; Vecchio, E. A.; Nguyen, A. T. N.; Sexton, P. M.; White, P. J.; May, L. T.; Christopoulos, A.; Scammells, P. J. Novel Irreversible Agonists Acting at the A1 Adenosine Receptor. J. Med. Chem. 2016, 59 (24), 11182– 11194, DOI: 10.1021/acs.jmedchem.6b01561[ACS Full Text], [CAS], Google Scholar274https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFynt77F&md5=6157d0f8b0b980dedf4462ddd263a807Novel Irreversible Agonists Acting at the A1 Adenosine ReceptorJorg, Manuela; Glukhova, Alisa; Abdul-Ridha, Alaa; Vecchio, Elizabeth A.; Nguyen, Anh T. N.; Sexton, Patrick M.; White, Paul J.; May, Lauren T.; Christopoulos, Arthur; Scammells, Peter J.Journal of Medicinal Chemistry (2016), 59 (24), 11182-11194CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The A1 adenosine receptor (A1AR) is an important G protein-coupled receptor that regulates a range of physiol. functions. Herein we report the discovery of novel irreversible agonists acting at the A1AR, which have the potential to serve as useful research tools for studying receptor structure and function. A series of novel adenosine derivs. bearing electrophilic substituents was synthesized, and four compds., 8b, 15a, 15b, and 15d, were shown to possess similar potency and efficacy to the ref. high efficacy agonist, NECA, in an assay of ERK1/2 phosphorylation assay. Insensitivity to antagonist addn. in a real-time, label-free, xCELLigence assay was subsequently used to identify compds. that likely mediated their agonism through an irreversible interaction with the A1AR. Of these compds., 15b and 15d were more directly validated as irreversible agonists of the A1AR using membrane-based [3H]DPCPX and [35S]GTPγS binding expts.
- 275Yang, X.; Dong, G.; Michiels, T. J. M.; Lenselink, E. B.; Heitman, L.; Louvel, J.; IJzerman, A. P. A Covalent Antagonist for the Human Adenosine A2A Receptor. Purinergic Signalling 2017, 13 (2), 191– 201, DOI: 10.1007/s11302-016-9549-9[Crossref], [PubMed], [CAS], Google Scholar275https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVSrtLrN&md5=7ce9f2fd7be76d83997cd3aac8eb91c1A covalent antagonist for the human adenosine A2A receptorYang, Xue; Dong, Guo; Michiels, Thomas J. M.; Lenselink, Eelke B.; Heitman, Laura; Louvel, Julien; IJzerman, Ad P.Purinergic Signalling (2017), 13 (2), 191-201CODEN: PSUIA9; ISSN:1573-9538. (Springer)The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallog. with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template mol. served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out expts. was set up to investigate the efficacy of covalent binding of LUF7445. All these expts. led us to conclude LUF7445 is a valuable mol. tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.
- 276Beauglehole, A. R.; Baker, S. P.; Scammells, P. J. Fluorosulfonyl-Substituted Xanthines as Selective Irreversible Antagonists for the A1-Adenosine Receptor. J. Med. Chem. 2000, 43 (26), 4973– 4980, DOI: 10.1021/jm000181f[ACS Full Text], [CAS], Google Scholar276https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXotFymurw%253D&md5=cf1fdfea7ede2f1f5874d0bd82ff9b38Fluorosulfonyl-Substituted Xanthines as Selective Irreversible Antagonists for the A1-Adenosine ReceptorBeauglehole, Anthony R.; Baker, Stephen P.; Scammells, Peter J.Journal of Medicinal Chemistry (2000), 43 (26), 4973-4980CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)FSCPX (1) has been reported to be a potent, selective, and irreversible antagonist for the A1-adenosine receptor (AR). To obtain an irreversible A1AR antagonist with potentially better stability and to further elucidate the effects of linker structure on the pharmacol. characteristics, several new analogs were targeted in which the labile ester linkage of 1 was replaced by more stable functionalities. In particular, alkyl and amide linkers between the xanthine pharmacophore and the reactive 4-fluorosulfonylphenyl group were explored. The data showed that the chem. compn. of the linker affects the affinity and apparent irreversible binding to the A1AR. Overall, compd. (I) appeared to have the most advantageous characteristics as a potential irreversible ligand for the A1AR. These include relatively high affinity for the A1AR as compared to the A2AAR, concn.-dependent and selective apparent irreversible binding to the A1AR, and ease of removal of unbound ligand from biol. membranes. These properties indicate that I has the potential to be a useful tool for further study of the structure and function of the A1AR.
- 277Glukhova, A.; Thal, D. M.; Nguyen, A. T.; Vecchio, E. A.; Jörg, M.; Scammells, P. J.; May, L. T.; Sexton, P. M.; Christopoulos, A. Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity. Cell 2017, 168, 867– 877, DOI: 10.1016/j.cell.2017.01.042[Crossref], [PubMed], [CAS], Google Scholar277https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjvVWrsrg%253D&md5=a5ff676cfedd6e11a899f65555a1ba62Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype SelectivityGlukhova, Alisa; Thal, David M.; Nguyen, Anh T.; Vecchio, Elizabeth A.; Jorg, Manuela; Scammells, Peter J.; May, Lauren T.; Sexton, Patrick M.; Christopoulos, ArthurCell (Cambridge, MA, United States) (2017), 168 (5), 867-877.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The adenosine A1 receptor (A1-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We detd. a 3.2 Å crystal structure of the A1-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A2A receptor (A2A-AR) structure. Mutational and computational anal. of A1-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a mol. basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.
- 278Kitz, R.; Wilson, I. B. Esters of Methanesulfonic Acid as Irreversible Inhibitors of Acetylcholinesterase. J. Biol. Chem. 1962, 237, 3245– 3249[PubMed], [CAS], Google Scholar278https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF38Xks1Cmsrg%253D&md5=f3993cf79ebb66299fada0ad04b15860Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesteraseKitz, R.; Wilson, Irwin B.Journal of Biological Chemistry (1962), 237 (), 3245-49CODEN: JBCHA3; ISSN:0021-9258.Nine esters (anhydrides) of methanesulfonic acid, analogs to well known carbamate (e.g. neostigmine) and phosphate (e.g. paraoxon) inhibitors, were examd. as possible irreversible inhibitors of acetylcholinesterase. Five were active. Of these, 4 formed initial reversible complexes. The progressive development of irreversible inhibition was slowed by reversible inhibitors, indicating that the active site is involved. The inhibited enzyme is not reactivated by water or by hydroxylamine, hydrazine, acetate, or pyridine-2-aldoxime methiodide. It is reactivated by pyridine-3-aldoxime methiodide. On the basis of the evidence presented, it may be concluded safely that the active methanesulfonates form methanesulfonyl enzyme derivs. and, therefore, belong to the class of acid-transferring or oxydiaphoric inhibitors.
- 279Moss, D. E.; Berlanga, P.; Hagan, M. M.; Sandoval, H.; Ishida, C. Methanesulfonyl Fluoride (MSF): A Double-Blind, Placebo-Controlled Study of Safety and Efficacy in the Treatment of Senile Dementia of the Alzheimer Type. Alzheimer Dis. Assoc. Disord. 1999, 13 (1), 20, DOI: 10.1097/00002093-199903000-00003[Crossref], [PubMed], [CAS], Google Scholar279https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXitVeqtL4%253D&md5=64f9fda8b3410f6d17ce7ba4704785cdMethanesulfonyl fluoride (MSF): a double-blind, placebo-controlled study of safety and efficacy in the treatment of senile dementia of the Alzheimer typeMoss, D. E.; Berlanga, P.; Hagan, M. M.; Sandoval, H.; Ishida, C.Alzheimer Disease and Associated Disorders (1999), 13 (1), 20-25CODEN: ADADE2; ISSN:0893-0341. (Lippincott Williams & Wilkins)The purpose of the present study was to evaluate methanesulfonyl fluoride (MSF), a very long-acting CNS-selective acetylcholinesterase (AChE) inhibitor, as a palliative treatment for senile dementia of the Alzheimer type (SDAT). In expt. I, MSF (0.03-0.18 mg/kg) was administered orally to 10 normal volunteers to measure toxicity and establish dose/response function in erythrocyte AChE. MSF produced a dose-response function of %inhibition = (40)(Log10[MSF mg/kg] + 51.7) with no toxicity at these doses. Expt. II was a 16-wk double-blind, placebo-controlled study of the safety and efficacy of MSF in doses of up to 0.18 mg/kg given three times per wk in 5 men and 10 women (60-82 yr), with Mini-Mental State Examn. (MMSE) scores of 9-24, who had SDAT. MSF produced a mean of 89.5% inhibition of erythrocyte AChE in patients and improved cognitive performance as measured by the MMSE, Alzheimer Disease Assessment Scale-Cognitive Subscale (ADAS-COG), Global Deterioration Scale, and the Clin. Interview Based Impression of Change (CIBIC). Most of the improvement on the ADAS-COG was maintained 8 wk after ending MSF. No patients left the study because of drug-related adverse events and there were no toxic effects. MSF may be a safe and effective palliative treatment for SDAT and further clin. trials in larger groups of patients are warranted.
- 280Moss, D. E.; Fariello, R. G.; Sahlmann, J.; Sumaya, I.; Pericle, F.; Braglia, E. A Randomized Phase I Study of Methanesulfonyl Fluoride, an Irreversible Cholinesterase Inhibitor, for the Treatment of Alzheimer’s Disease. Br. J. Clin. Pharmacol. 2013, 75 (5), 1231– 1239, DOI: 10.1111/bcp.12018[Crossref], [PubMed], [CAS], Google Scholar280https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXps1CqsLo%253D&md5=ef0e3ba992302e53d74ade7647d8554cA randomized phase I study of methanesulfonyl fluoride, an irreversible cholinesterase inhibitor, for the treatment of Alzheimer's diseaseMoss, Donald E.; Fariello, Ruggero G.; Sahlmann, Jorg; Sumaya, Isabel; Pericle, Federica; Braglia, EnricoBritish Journal of Clinical Pharmacology (2013), 75 (5), 1231-1239CODEN: BCPHBM; ISSN:1365-2125. (Wiley-Blackwell)AIMS: To ascertain the tolerability profile of single and repeated oral doses of methanesulfonyl fluoride (MSF, SNX-001) in healthy aged subjects, and to det. the degree of erythrocyte acetylcholinesterase (AChE) inhibition induced by MSF after single and repeated oral doses. METHODS: To calc. properly the kinetics and the duration of AChE inhibition, the effects of MSF were also studied in rodents. These expts. suggested that MSF administered three times per wk should provide safe and efficacious AChE inhibition. In a randomized placebo-controlled phase I study, 3.6 mg, 7.2 mg or 10.8 mg MSF were then orally administered to 27 consenting healthy volunteers (aged 50 to 72 years). After a single dose phase and a 1 wk wash-out period, the subjects received the same doses three times per wk for 2 wk. RESULTS: Twenty-two out of the 27 subjects completed the study. Four patients withdrew due to adverse events (AEs) and one for non-compliance. Erythrocyte AChE was inhibited by a total of 33%, 46%, and 62% after 2 wk of 3.6 mg, 7.2 mg and 10.8 mg MSF, resp. No serious AEs occurred. The most frequent AEs were headache (27%), nausea (11%) and diarrhea (8%). CONCLUSIONS: MSF proved to be well tolerated even with repeated oral dosing. It is estd. that MSF provided a degree of AChE inhibition that should effectively enhance memory. This mol. deserves to be tested for efficacy in a pilot randomized controlled study in patients with Alzheimer's disease.
- 281Jones, L. H. Reactive Chemical Probes: Beyond the Kinase Cysteinome. Angew. Chem., Int. Ed. 2018, 57 (30), 9220– 9223, DOI: 10.1002/anie.201802693[Crossref], [CAS], Google Scholar281https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFaltLbF&md5=3aa3b629e71ea55d7627c2a94da895efReactive Chemical Probes: Beyond the Kinase CysteinomeJones, Lyn H.Angewandte Chemie, International Edition (2018), 57 (30), 9220-9223CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The reaction of small-mol. chem. probes with proteins has been harnessed to develop covalent inhibitor drugs and protein-profiling technologies. This Essay discusses some of the recent enhancements to the chem. biol. toolkit that are enabling the study of previously uncharted areas of chemoproteomic space. An anal. of the kinome is used to illustrate the potential for these approaches enable the pursuit of new targets using reactive chem. probes.
- 282Mukherjee, H.; Debreczeni, J.; Breed, J.; Tentarelli, S.; Aquila, B.; Dowling, J. E.; Whitty, A.; Grimster, N. P. A Study of the Reactivity of S(VI)–F Containing Warheads with Nucleophilic Amino-Acid Side Chains under Physiological Conditions. Org. Biomol. Chem. 2017, 15 (45), 9685– 9695, DOI: 10.1039/C7OB02028G[Crossref], [PubMed], [CAS], Google Scholar282https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslSgsbnI&md5=cf00897a4b0b184e2d68df498ec8cbb7A study of the reactivity of S(VI)-F containing warheads with nucleophilic amino-acid side chains under physiological conditionsMukherjee, H.; Debreczeni, J.; Breed, J.; Tentarelli, S.; Aquila, B.; Dowling, J. E.; Whitty, A.; Grimster, N. P.Organic & Biomolecular Chemistry (2017), 15 (45), 9685-9695CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Sulfonyl fluorides (SFs) have recently emerged as a promising warhead for the targeted covalent modification of proteins. Despite numerous examples of the successful deployment of SFs as covalent probe compds., a detailed exploration of the factors influencing the stability and reactivity of SFs has not yet appeared. In this work we present an extensive study on the influence of steric and electronic factors on the reactivity and stability of the SF and related SVI-F groups. While SFs react rapidly with N-acetylcysteine, the resulting adducts were found to be unstable, rendering SFs inappropriate for the durable covalent inhibition of cysteine residues. In contrast, SFs afforded stable adducts with both N-acetyltyrosine and N-acetyllysine; furthermore, we show that the reactivity of arylsulfonyl fluorides towards these nucleophilic amino acids can be predictably modulated by adjusting the electronic properties of the warhead. These trends were largely conserved when the covalent reaction occurred within a protein binding pocket. We have also obtained a crystal structure depicting covalent modification of the catalytic lysine of a tyrosine kinase (FGFR1) by the ATP analog 5'-O-3-((fluorosulfonyl)benzoyl)adenosine (m-FSBA). Highly reactive warheads were demonstrated to be unstable with respect to hydrolysis in buffered aq. solns., indicating that warhead reactivity must be carefully tuned to provide optimal rates of protein modification. Our results demonstrate that the reactivity of SFs complements that of more commonly studied acrylamides, and we hope that this work spurs the rational design of novel SF-contg. covalent probe compds. and inhibitors, particularly in cases where a suitably positioned cysteine residue is not present.
- 283Lundblad, R. L. Chemical Reagents for Protein Modification, 4th ed.; CRC Press: Boca Raton, 2017.
- 284Chinthakindi, P. K.; Arvidsson, P. I. Sulfonyl Fluorides (SFs): More Than Click Reagents?. Eur. J. Org. Chem. 2018, 2018 (27–28), 3648– 3666, DOI: 10.1002/ejoc.201800464[Crossref], [CAS], Google Scholar284https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1yktL%252FE&md5=819292301bdb81cab0f12f4c535d8078Sulfonyl Fluorides (SFs): More Than Click Reagents?Chinthakindi, Praveen K.; Arvidsson, Per I.European Journal of Organic Chemistry (2018), 2018 (27-28), 3648-3666CODEN: EJOCFK; ISSN:1099-0690. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Sulfonyl fluoride (SF)-contg. substances are currently attracting enormous attention among practitioners of both chem. biol. and synthetic org. chem. The groups of Jones and Liskamp have demonstrated the potential of sulfonyl fluorides as selective covalent inhibitors in studies related to drug discovery and chem. biol., resp., in the last few years. The Sharpless group has extended the repertoire of "click-reactions" to those involving sulfonyl fluorides, i.e., sulfur-fluoride exchange (SuFEx), a development that quickly triggered the interest in this functional group in the community of synthetic org. chemists. In this microreview, we aim to give an account of the synthetic chem. surrounding sulfonyl fluoride contg. substances from a historical perspective to present day developments.
- 285Wang, N.; Yang, B.; Fu, C.; Zhu, H.; Zheng, F.; Kobayashi, T.; Liu, J.; Li, S.; Ma, C.; Wang, P. G.; Wang, Q.; Wang, L. Genetically Encoding Fluorosulfate-l-Tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo. J. Am. Chem. Soc. 2018, 140 (15), 4995– 4999, DOI: 10.1021/jacs.8b01087[ACS Full Text], [CAS], Google Scholar285https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmslGqs78%253D&md5=30583248ed49b2499ef4fd1481ac5adfGenetically Encoding Fluorosulfate-L-tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in VivoWang, Nanxi; Yang, Bing; Fu, Caiyun; Zhu, He; Zheng, Feng; Kobayashi, Tomonori; Liu, Jun; Li, Shanshan; Ma, Cheng; Wang, Peng G.; Wang, Qian; Wang, LeiJournal of the American Chemical Society (2018), 140 (15), 4995-4999CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Introducing new chem. reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering in vivo. Latent bioreactive unnatural amino acids (Uaas) can be incorporated into proteins to react with target natural amino acid residues via proximity-enabled reactivity. To expand the diversity of proteins amenable to such reactivity in vivo, a chem. functionality that is biocompatible and able to react with multiple natural residues under physiol. conditions is highly desirable. Here the authors report the genetic encoding of fluorosulfate-L-tyrosine (FSY), the first latent bioreactive Uaa that undergoes sulfur-fluoride exchange (SuFEx) on proteins in vivo. FSY was found nontoxic to Escherichia coli and mammalian cells; after being incorporated into proteins, it selectively reacted with proximal lysine, histidine, and tyrosine via SuFEx, generating covalent intraprotein bridge and interprotein crosslink of interacting proteins directly in living cells. The proximity-activatable reactivity, multitargeting ability, and excellent biocompatibility of FSY will be invaluable for covalent manipulation of proteins in vivo. Moreover, genetically encoded FSY hereby empowers general proteins with the next generation of click chem., SuFEx, which will afford broad utilities in chem. biol., drug discovery, and biotherapeutics.
- 286Zhao, Q.; Ouyang, X.; Wan, X.; Gajiwala, K. S.; Kath, J. C.; Jones, L. H.; Burlingame, A. L.; Taunton, J. Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes. J. Am. Chem. Soc. 2017, 139 (2), 680– 685, DOI: 10.1021/jacs.6b08536[ACS Full Text], [CAS], Google Scholar286https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslWisA%253D%253D&md5=3f643a1b32c83fa05932f42b54e96e47Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride ProbesZhao, Qian; Ouyang, Xiaohu; Wan, Xiaobo; Gajiwala, Ketan S.; Kath, John C.; Jones, Lyn H.; Burlingame, Alma L.; Taunton, JackJournal of the American Chemical Society (2017), 139 (2), 680-685CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallog. and mass spectrometry (MS) confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concns. of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clin. relevant concns., highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications.
- 287Becher, I.; Savitski, M. M.; Savitski, M. F.; Hopf, C.; Bantscheff, M.; Drewes, G. Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP. ACS Chem. Biol. 2013, 8 (3), 599– 607, DOI: 10.1021/cb3005879[ACS Full Text], [CAS], Google Scholar287https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhslyjsL7P&md5=a9c8ac0cfbdcda3f20f2d0d91422374fAffinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTPBecher, Isabelle; Savitski, Mikhail M.; Savitski, Maria Faelth; Hopf, Carsten; Bantscheff, Marcus; Drewes, GerardACS Chemical Biology (2013), 8 (3), 599-607CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Most kinase inhibitor drugs target the binding site of the nucleotide cosubstrate ATP. The high intracellular concn. of ATP can strongly affect inhibitor potency and selectivity depending on the affinity of the target kinase for ATP. Here we used a defined chemoproteomics system based on competition-binding assays in cell exts. from Jurkat and SK-MEL-28 cells with immobilized ATP mimetics (kinobeads). This system enabled us to assess the affinities of more than 200 kinases for the cellular nucleotide cofactors ATP, ADP, and GTP and the effects of the divalent metal ions Mg2+ and Mn2+. The affinity values detd. in this system were largely consistent across the two cell lines, indicating no major dependence on kinase expression levels. Kinase-ATP affinities range from low micromolar to millimolar, which has profound consequences for the prediction of cellular effects from inhibitor selectivity profiles. Only a small no. of kinases including CK2, MEK, and BRAF exhibited affinity for GTP. This extensive and consistent data set of kinase-nucleotide affinities, detd. for native enzymes under defined exptl. conditions, will represent a useful resource for kinase drug discovery.
- 288Baranczak, A.; Liu, Y.; Connelly, S.; Du, W.-G. H.; Greiner, E. R.; Genereux, J. C.; Wiseman, R. L.; Eisele, Y. S.; Bradbury, N. C.; Dong, J.; Noodleman, L.; Sharpless, K. B.; Wilson, I. A.; Encalada, S. E.; Kelly, J. W. A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis Elegans. J. Am. Chem. Soc. 2015, 137 (23), 7404– 7414, DOI: 10.1021/jacs.5b03042[ACS Full Text], [CAS], Google Scholar288https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVans7bN&md5=ba307289df4c28c469905a8250d11126A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis elegansBaranczak, Aleksandra; Liu, Yu; Connelly, Stephen; Du, Wen-Ge Han; Greiner, Erin R.; Genereux, Joseph C.; Wiseman, R. Luke; Eisele, Yvonne S.; Bradbury, Nadine C.; Dong, Jiajia; Noodleman, Louis; Sharpless, K. Barry; Wilson, Ian A.; Encalada, Sandra E.; Kelly, Jeffery W.Journal of the American Chemical Society (2015), 137 (23), 7404-7414CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Fluorogenic probes, due to their often greater spatial and temporal sensitivity in comparison to permanently fluorescent small mols., represent powerful tools to study protein localization and function in the context of living systems. Herein, we report fluorogenic probe 4, a 1,3,4-oxadiazole designed to bind selectively to transthyretin (TTR). Probe 4 comprises a fluorosulfate group not previously used in an environment-sensitive fluorophore. The fluorosulfate functional group does not react covalently with TTR on the time scale required for cellular imaging, but does red shift the emission max. of probe 4 in comparison to its nonfluorosulfated analog. We demonstrate that probe 4 is dark in aq. buffers, whereas the TTR·4 complex exhibits a fluorescence emission max. at 481 nm. The addn. of probe 4 to living HEK293T cells allows efficient binding to and imaging of exogenous TTR within intracellular organelles, including the mitochondria and the endoplasmic reticulum. Furthermore, live Caenorhabditis elegans expressing human TTR transgenically and treated with probe 4 display TTR·4 fluorescence in macrophage-like coelomocytes. An analog of fluorosulfate probe 4 does react selectively with TTR without labeling the remainder of the cellular proteome. Studies on this analog suggest that certain aryl fluorosulfates, due to their cell and organelle permeability and activatable reactivity, could be considered for the development of protein-selective covalent probes.
- 289Grimster, N. P.; Connelly, S.; Baranczak, A.; Dong, J.; Krasnova, L. B.; Sharpless, K. B.; Powers, E. T.; Wilson, I. A.; Kelly, J. W. Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis While Affording a Fluorescent Conjugate. J. Am. Chem. Soc. 2013, 135 (15), 5656– 5668, DOI: 10.1021/ja311729d[ACS Full Text], [CAS], Google Scholar289https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFejsrs%253D&md5=d019c851c9fcdc108a8de6af617e1b5bAromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis while Affording a Fluorescent ConjugateGrimster, Neil P.; Connelly, Stephen; Baranczak, Aleksandra; Dong, Jiajia; Krasnova, Larissa B.; Sharpless, K. Barry; Powers, Evan T.; Wilson, Ian A.; Kelly, Jeffery W.Journal of the American Chemical Society (2013), 135 (15), 5656-5668CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Mols. that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) was conceived of by structure-based design principles and was chem. synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ∼1400 times faster than the hydrolysis reaction outside the thyroxine binding site. X-ray crystallog. confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. Eleven of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. Eleven of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore.
- 290Dalton, S. E.; Dittus, L.; Thomas, D. A.; Convery, M. A.; Nunes, J.; Bush, J. T.; Evans, J. P.; Werner, T.; Bantscheff, M.; Murphy, J. A.; Campos, S. Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition. J. Am. Chem. Soc. 2018, 140 (3), 932– 939, DOI: 10.1021/jacs.7b08979[ACS Full Text], [CAS], Google Scholar290https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFKitrrK&md5=90bb399303a942fb87f1b9096eb8bdc6Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase InhibitionDalton, Samuel E.; Dittus, Lars; Thomas, Daniel A.; Convery, Maire A.; Nunes, Joao; Bush, Jacob T.; Evans, John P.; Werner, Thilo; Bantscheff, Marcus; Murphy, John A.; Campos, SebastienJournal of the American Chemical Society (2018), 140 (3), 932-939CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to date in the development of chem. probes and approved drugs. However, this approach is limited to ∼200 kinases possessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing selective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compd. shows high levels of selectivity over a selection of lipid and protein kinases in biochem. assays, as well as covalent binding to very few off-target proteins in live-cell proteomic studies. We anticipate this approach could offer a general strategy, as an alternative to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.
- 291Gupta, R. C.; Sachana, M.; Mukherjee, I. M.; Doss, R. B.; Malik, J. K.; Milatovic, D. Organophosphates and Carbamates. In Veterinary Toxicology; Gupta, R. C., Ed.; Academic Press, 2018; pp. 495– 508, DOI: 10.1016/B978-0-12-811410-0.00037-4 .
- 292Tamura, T.; Ueda, T.; Goto, T.; Tsukidate, T.; Shapira, Y.; Nishikawa, Y.; Fujisawa, A.; Hamachi, I. Rapid Labelling and Covalent Inhibition of Intracellular Native Proteins Using Ligand-Directed N -Acyl- N -Alkyl Sulfonamide. Nat. Commun. 2018, 9 (1), 1870, DOI: 10.1038/s41467-018-04343-0[Crossref], [PubMed], [CAS], Google Scholar292https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MfjtFygug%253D%253D&md5=98b2280e760b0e170af05a6283c5d794Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamideTamura Tomonori; Ueda Tsuyoshi; Goto Taiki; Tsukidate Taku; Nishikawa Yuki; Fujisawa Alma; Hamachi Itaru; Shapira Yonatan; Hamachi ItaruNature communications (2018), 9 (1), 1870 ISSN:.Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~10(4) M(-1) s(-1), comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.
- 293Evans, M. J.; Saghatelian, A.; Sorensen, E. J.; Cravatt, B. F. Target Discovery in Small-Molecule Cell-Based Screens by in Situ Proteome Reactivity Profiling. Nat. Biotechnol. 2005, 23, 1303, DOI: 10.1038/nbt1149[Crossref], [PubMed], [CAS], Google Scholar293https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVOhu7nP&md5=1fc3d093fe8c7c7ccd7d610f10df105cTarget discovery in small-molecule cell-based screens by in situ proteome reactivity profilingEvans, Michael J.; Saghatelian, Alan; Sorensen, Erik J.; Cravatt, Benjamin F.Nature Biotechnology (2005), 23 (10), 1303-1307CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Chem. genomics aims to discover small mols. that affect biol. processes through the perturbation of protein function. However, detg. the protein targets of bioactive compds. remains a formidable challenge. The authors address this problem here through the creation of a natural product-inspired small-mol. library bearing protein-reactive elements. Cell-based screening identified a compd., MJE3 (I), that inhibits breast cancer cell proliferation. In situ proteome reactivity profiling revealed that MJE3, but not other library members, covalently labeled the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), resulting in enzyme inhibition. Interestingly, MJE3 labeling and inhibition of PGAM1 were obsd. exclusively in intact cells. These results support the hypothesis that cancer cells depend on glycolysis for viability and promote PGAM1 as a potential therapeutic target. More generally, the incorporation of protein-reactive compds. into chem. genomics screens offers a means to discover targets of bioactive small mols. in living systems, thereby enabling downstream mechanistic investigations.
- 294Evans, M. J.; Morris, G. M.; Wu, J.; Olson, A. J.; Sorensen, E. J.; Cravatt, B. F. Mechanistic and Structural Requirements for Active Site Labeling of Phosphoglycerate Mutase by Spiroepoxides. Mol. BioSyst. 2007, 3, 495– 506, DOI: 10.1039/b705113a[Crossref], [PubMed], [CAS], Google Scholar294https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXms1Wnt74%253D&md5=b69570bc329ddc6db39235b8c241d6c7Mechanistic and structural requirements for active site labeling of phosphoglycerate mutase by spiroepoxidesEvans, Michael J.; Morris, Garrett M.; Wu, Jane; Olson, Arthur J.; Sorensen, Erik J.; Cravatt, Benjamin F.Molecular BioSystems (2007), 3 (7), 495-506CODEN: MBOIBW; ISSN:1742-206X. (Royal Society of Chemistry)We recently reported the pharmacol. screening of a natural products-inspired library of spiroepoxide probes, resulting in the discovery of an agent MJE3 that displayed anti-proliferative effects in human breast cancer cells. MJE3 was found to covalently inactivate phosphoglycerate mutase-1 (PGAM1), a glycolytic enzyme with postulated roles in cancer cell metab. and proliferation. Considering that MJE3 is one of the first examples of a cell-permeable, small-mol. inhibitor for PGAM1, we pursued a detailed examn. of its mechanism and structural requirements for covalent inactivation. MJE3 was found to label PGAM1 on lysine-100, a conserved active site residue implicated in substrate recognition. Structural features of MJE3 important for PGAM1 labeling included two key recognition elements (an indole ring and carboxylic acid), the stereochem. orientation of the spiroepoxide, and presentation of these various binding/reactive groups on a rigid cyclohexane scaffold. Modeling studies of the docked MJE3-PGAM1 complex provide a structural rationale for these stringent requirements. Overall, these studies indicate that a special combination of binding and reactive elements are united in the MJE3 structure to inactivate PGAM1. More generally, our findings provide further evidence that useful pharmacol. tools can emerge from screening structurally diverse libraries of protein-reactive probes.
- 295Bongard, J.; Lorenz, M.; Vetter, I. R.; Stege, P.; Porfetye, A. T.; Schmitz, A. L.; Kaschani, F.; Wolf, A.; Koch, U.; Nussbaumer, P.; Klebl, B.; Kaiser, M.; Ehrmann, M. Identification of Noncatalytic Lysine Residues from Allosteric Circuits via Covalent Probes. ACS Chem. Biol. 2018, 13 (5), 1307– 1312, DOI: 10.1021/acschembio.8b00101[ACS Full Text], [CAS], Google Scholar295https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnsFGisLo%253D&md5=b791163873aa49010b449540aab5be32Identification of Noncatalytic Lysine Residues from Allosteric Circuits via Covalent ProbesBongard, Jens; Lorenz, Marian; Vetter, Ingrid R.; Stege, Patricia; Porfetye, Arthur T.; Schmitz, Anna Laura; Kaschani, Farnusch; Wolf, Alex; Koch, Uwe; Nussbaumer, Peter; Klebl, Bert; Kaiser, Markus; Ehrmann, MichaelACS Chemical Biology (2018), 13 (5), 1307-1312CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Covalent modifications of nonactive site lysine residues by small mol. probes has recently evolved into an important strategy for interrogating biol. systems. Here, we report the discovery of a class of bioreactive compds. that covalently modify lysine residues in DegS, the rate limiting protease of the essential bacterial outer membrane stress response pathway. These modifications lead to an allosteric activation and allow the identification of novel residues involved in the allosteric activation circuit. These findings were validated by structural analyses via X-ray crystallog. and cell-based reporter systems. We anticipate that our findings are not only relevant for a deeper understanding of the structural basis of allosteric activation in DegS and other HtrA serine proteases but also pinpoint an alternative use of covalent small mols. for probing essential biochem. mechanisms.
- 296Diethelm, S.; Schafroth, M. A.; Carreira, E. M. Amine-Selective Bioconjugation Using Arene Diazonium Salts. Org. Lett. 2014, 16 (15), 3908– 3911, DOI: 10.1021/ol5016509[ACS Full Text], [CAS], Google Scholar296https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFCrtbfF&md5=91876c83f67d31189c501b567630900eAmine-Selective Bioconjugation Using Arene Diazonium SaltsDiethelm, Stefan; Schafroth, Michael A.; Carreira, Erick M.Organic Letters (2014), 16 (15), 3908-3911CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)A novel bioconjugation strategy is presented that relies on the coupling of diazonium terephthalates with amines in proteins. The diazonium captures the amine while the vicinal ester locks it through cyclization, ensuring no reversibility. The reaction is highly efficient and proceeds under mild conditions and short reaction times. Densely functionalized, complex natural products were directly coupled to proteins using low concns. of coupling partners.
- 297Tung, C. L.; Wong, C. T. T.; Fung, E. Y. M.; Li, X. Traceless and Chemoselective Amine Bioconjugation via Phthalimidine Formation in Native Protein Modification. Org. Lett. 2016, 18 (11), 2600– 2603, DOI: 10.1021/acs.orglett.6b00983[ACS Full Text], [CAS], Google Scholar297https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XotFelsLs%253D&md5=93516d763bd6399a7753e7e5931d77e1Traceless and chemoselective amine bioconjugation via phthalimidine formation in native protein modificationTung, Chun Ling; Wong, Clarence T. T.; Fung, Eva Yi Man; Li, XuechenOrganic Letters (2016), 18 (11), 2600-2603CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)Ortho-Phthalaldehyde (OPA) and its derivs. are found to react chemoselectively with amino groups on peptides and proteins rapidly and tracelessly under the physiol. condition via formation of phthalimidines, which provides a novel and promising approach when performing bioconjugation on native proteins. The notable advantages of this method over the existing native protein lysine-labeling approaches include a traceless process, a self-reacting, specific and fast reaction, ease of operation, and the ability to use nonhydrolyzable reagents. Its applications have been effectively demonstrated including conjugation of peptides and proteins, and generation of an active PEGlyated L-asparaginase.
- 298Ritter, E.; Przybylski, P.; Brzezinski, B.; Bartl, F. Schiff Bases in Biological Systems. Curr. Org. Chem. 2009, 13 (3), 241– 249, DOI: 10.2174/138527209787314805[Crossref], [CAS], Google Scholar298https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXkslCnu7Y%253D&md5=d09e6ac15bea3a2959af966aef7bc7feSchiff bases in biological systemsRitter, Eglof; Przybylski, Piotr; Brzezinski, Bogumil; Bartl, FranzCurrent Organic Chemistry (2009), 13 (3), 241-249CODEN: CORCFE; ISSN:1385-2728. (Bentham Science Publishers Ltd.)A review. In many cases, the cofactors of biol. important proteins are linked to their apoproteins by Schiff bases. In this review, we discuss three classes of proteins in which Schiff bases are crucial for their function and catalytic mechanisms. First, we focus on retinylidene proteins, in which the cofactor retinal is fixed to the apoprotein by a protonated Schiff base. Light-induced retinal isomerization triggers a photocycle or a cascade of structural changes in which deprotonation of the Schiff base is a crucial step. We compare the photocycles of archaeal type rhodopsins, channelrhodopsins and the light induced cascades of visual rhodopsins. Pyridoxal phosphate (PLP) dependent enzymes are summarized in the second section. In these proteins, the cofactor PLP is linked via a Schiff base to the side chain of a lysine. In most cases, hydrolysis of this linkage is crit. for substrate binding. Finally, aldolases, that catalyze aldol cleavage during glycolysis, contain a lysine residue at their active center. Formation of a protonated Schiff base linkage to a carbonyl group of the substrate is the first step of the catalytic mechanism. This will be discussed in the last section.
- 299Malátková, P.; Wsól, V. Carbonyl Reduction Pathways in Drug Metabolism. Drug Metab. Rev. 2014, 46 (1), 96– 123, DOI: 10.3109/03602532.2013.853078[Crossref], [PubMed], [CAS], Google Scholar299https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVyksr8%253D&md5=9e605b6554b02cfb016c191128ba9e67Carbonyl reduction pathways in drug metabolismMalatkova, Petra; Wsol, VladimirDrug Metabolism Reviews (2014), 46 (1), 96-123CODEN: DMTRAR; ISSN:0360-2532. (Informa Healthcare)A review. The understanding of drug biotransformation is an important medical topic. The oxidative pathways that involve CYPs have been extensively studied in drug metab. in contrast to the reductive pathways. This review focuses on drugs that have been reported to be reduced at the carbonyl group in vivo. Although the carbonyl redn. of these drugs is well known, our understanding of the carbonyl reducing enzymes (CRE) that perform these reactions is limited. We have summarized the published data in order to thoroughly describe the reductive metab. of the selected drugs and to demonstrate the role of carbonyl redn. in the context of their overall metab. The no. of drugs recognized as substrates for CREs has increased considerably in recent years. Moreover, the importance of carbonyl redn. in the overall metab. of these drugs is often surprisingly high. Because only limited information is available about the CREs responsible for these reactions, addnl. research is needed to improve our understanding of the metab. of drugs undergoing carbonyl redn. Carbonyl redn. should be investigated during drug development because it can either pos. or neg. influence drug efficacy.
- 300Cal, P. M. S. D.; Vicente, J. B.; Pires, E.; Coelho, A. V.; Veiros, L. F.; Cordeiro, C.; Gois, P. M. P. Iminoboronates: A New Strategy for Reversible Protein Modification. J. Am. Chem. Soc. 2012, 134 (24), 10299– 10305, DOI: 10.1021/ja303436y[ACS Full Text], [CAS], Google Scholar300https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnsF2gurY%253D&md5=ad8e9263660d93401e1ccad61cd312dbIminoboronates: A new strategy for reversible protein modificationCal, Pedro M. S. D.; Vicente, Joao B.; Pires, Elisabete; Coelho, Ana V.; Veiros, Luis F.; Cordeiro, Carlos; Gois, Pedro M. P.Journal of the American Chemical Society (2012), 134 (24), 10299-10305CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein modification has entered the limelight of chem. and biol. sciences, since, by appending small mols. into proteins surfaces, fundamental biol. and biophys. processes may be studied and even modulated in a physiol. context. Herein the authors present a new strategy to modify the lysine's ε-amino group and the protein's N-terminal, based on the formation of stable iminoboronates in aq. media. This functionality enables the stable and complete modification of these amine groups, which can be reversible upon the addn. of fructose, dopamine, or glutathione. A detailed DFT study is also presented to rationalize the obsd. stability toward hydrolysis of the iminoboronate constructs.
- 301Adams, J.; Kauffman, M. Development of the Proteasome Inhibitor VelcadeTM (Bortezomib). Cancer Invest. 2004, 22 (2), 304– 311, DOI: 10.1081/CNV-120030218[Crossref], [PubMed], [CAS], Google Scholar301https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjtl2qtb0%253D&md5=6448e87e5e90eecf7399342bdbf50979Development of the Proteasome Inhibitor Velcade (Bortezomib)Adams, Julian; Kauffman, MichaelCancer Investigation (2004), 22 (2), 304-311CODEN: CINVD7; ISSN:0735-7907. (Marcel Dekker, Inc.)A review. The dipeptide boronic acid analog VELCADE (Bortezomib; formerly known as PS-341, LDP-341 and MLM341) is a potent and selective inhibitor of the proteasome, a multicatalytic enzyme that mediates many cellular regulatory signals by degrading regulatory proteins or their inhibitors. The proteasome is, thus, a potential target for pharmacol. agents. Bortezomib, the first proteasome inhibitor to reach clin. trials, has shown in vitro and in vivo activity against a variety of malignancies, including myeloma, chronic lymphocytic leukemia, prostate cancer, pancreatic cancer, and colon cancer. The drug is rapidly cleared from the vascular compartment, but a novel pharmacodynamic assay has shown that bortezomib-mediated proteasome blockade is dose-dependent and reversible. Based on phase I studies demonstrating that bortezomib has manageable toxicities in patients with advanced cancers, phase II trials have been initiated for both solid and hematol. malignancies.
- 302Bandyopadhyay, A.; McCarthy, K. A.; Kelly, M. A.; Gao, J. Targeting Bacteria via Iminoboronate Chemistry of Amine-Presenting Lipids. Nat. Commun. 2015, 6, 6561, DOI: 10.1038/ncomms7561[Crossref], [PubMed], [CAS], Google Scholar302https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXotlahsrY%253D&md5=c5e382b0d72f27f7911694d743322da9Targeting bacteria via iminoboronate chemistry of amine-presenting lipidsBandyopadhyay, Anupam; McCarthy, Kelly A.; Kelly, Michael A.; Gao, JianminNature Communications (2015), 6 (), 6561CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Synthetic mols. that target specific lipids serve as powerful tools for understanding membrane biol. and may also enable new applications in biotechnol. and medicine. For example, selective recognition of bacterial lipids may give rise to novel antibiotics, as well as diagnostic methods for bacterial infection. Currently known lipid-binding mols. primarily rely on noncovalent interactions to achieve lipid selectivity. Here we show that targeted recognition of lipids can be realized by selectively modifying the lipid of interest via covalent bond formation. Specifically, we report an unnatural amino acid that preferentially labels amine-presenting lipids via iminoboronate formation under physiol. conditions. By targeting phosphatidylethanolamine and lysylphosphatidylglycerol, the two lipids enriched on bacterial cell surfaces, the iminoboronate chem. allows potent labeling of Gram-pos. bacteria even in the presence of 10% serum, while bypassing mammalian cells and Gram-neg. bacteria. The covalent strategy for lipid recognition should be extendable to other important membrane lipids.
- 303Bandyopadhyay, A.; Gao, J. Iminoboronate Formation Leads to Fast and Reversible Conjugation Chemistry of α-Nucleophiles at Neutral PH. Chem. - Eur. J. 2015, 21 (42), 14748– 14752, DOI: 10.1002/chem.201502077[Crossref], [PubMed], [CAS], Google Scholar303https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVSis7zF&md5=03b9d05efc18ebe7534549609ccc5361Iminoboronate Formation Leads to Fast and Reversible Conjugation Chemistry of α-Nucleophiles at Neutral pHBandyopadhyay, Anupam; Gao, JianminChemistry - A European Journal (2015), 21 (42), 14748-14752CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)Bioorthogonal reactions that are fast and reversible under physiol. conditions are in high demand for biol. applications. Herein, an ortho boronic acid substituent makes aryl ketones rapidly conjugate with α-nucleophiles at neutral pH. Specifically, 2-acetylphenylboronic acid and derivs. conjugate with phenylhydrazine with rate consts. of 102 to 103 M-1s-1, comparable to the fastest bioorthogonal conjugations known to date. 11B NMR anal. revealed the varied extent of iminoboronate formation of the conjugates, in which the imine nitrogen forms a dative bond with boron. The iminoboronate formation activates the imines for hydrolysis and exchange, rendering these oxime/hydrazone conjugations reversible and dynamic under physiol. conditions. The fast and dynamic nature of the iminoboronate chem. should find wide applications in biol.
- 304Akçay, G.; Belmonte, M. A.; Aquila, B.; Chuaqui, C.; Hird, A. W.; Lamb, M. L.; Rawlins, P. B.; Su, N.; Tentarelli, S.; Grimster, N. P.; Su, Q. Inhibition of Mcl-1 through Covalent Modification of a Noncatalytic Lysine Side Chain. Nat. Chem. Biol. 2016, 12 (11), 931– 936, DOI: 10.1038/nchembio.2174[Crossref], [PubMed], [CAS], Google Scholar304https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKqtb3P&md5=bda5720579a9b5e6027632d2cffba2e1Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chainAkcay, Gizem; Belmonte, Matthew A.; Aquila, Brian; Chuaqui, Claudio; Hird, Alexander W.; Lamb, Michelle L.; Rawlins, Philip B.; Su, Nancy; Tentarelli, Sharon; Grimster, Neil P.; Su, QibinNature Chemical Biology (2016), 12 (11), 931-936CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Targeted covalent inhibition of disease-assocd. proteins has become a powerful methodol. in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here the authors used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to the knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chem. strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochem. and cell-based assays. The authors identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biol. phenomena.
- 305Harris, T. K.; Turner, G. J. Structural Basis of Perturbed PKa Values of Catalytic Groups in Enzyme Active Sites. IUBMB Life 2002, 53 (2), 85– 98, DOI: 10.1080/15216540211468[Crossref], [PubMed], [CAS], Google Scholar305https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XktlGisL8%253D&md5=fd5c17ecf362bb5d16790fbea204d512Structural basis of perturbed pKa values of catalytic groups in enzyme active sitesHarris, Thomas K.; Turner, George J.IUBMB Life (2002), 53 (2), 85-98CODEN: IULIF8; ISSN:1521-6543. (Taylor & Francis Inc.)A review with 81 refs. In protein and RNA macromols., only a limited no. of different side-chain chem. groups are available to function as catalysts. The myriad of enzyme-catalyzed reactions results from the ability of most of these groups to function either as nucleophilic, electrophilic, or general acid-base catalysts, and the key to their adapted chem. function lies in their states of protonation. Ionization is detd. by the intrinsic pKa of the group and the microenvironment created around the group by the protein or RNA structure, which perturbs its intrinsic pKa to its functional or apparent pKa. These pKa shifts result from interactions of the catalytic group with other fully or partially charged groups as well as the polarity or dielec. of the medium that surrounds it. The electrostatic interactions between ionizable groups found on the surface of macromols. are weak and cause only slight pKa perturbations (<2 units). The sum of many of these weak electrostatic interactions helps contribute to the stability of native or folded macromols. and their ligand complexes. However, the pKa values of catalytic groups that are found in the active sites of numerous enzymes are significantly more perturbed (>2 units) and are the subject of this review. The magnitudes of these pKa perturbations are analyzed with respect to the structural details of the active site microenvironment and the energetics of the reactions that they catalyze.
- 306Schwans, J. P.; Sunden, F.; Gonzalez, A.; Tsai, Y.; Herschlag, D. Uncovering the Determinants of a Highly Perturbed Tyrosine PKa in the Active Site of Ketosteroid Isomerase. Biochemistry 2013, 52 (44), 7840– 7855, DOI: 10.1021/bi401083b[ACS Full Text], [CAS], Google Scholar306https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFehu7vK&md5=2b02ad3af9f63850628edd6355beb7e1Uncovering the Determinants of a Highly Perturbed Tyrosine pKa in the Active Site of Ketosteroid IsomeraseSchwans, Jason P.; Sunden, Fanny; Gonzalez, Ana; Tsai, Yingssu; Herschlag, DanielBiochemistry (2013), 52 (44), 7840-7855CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Within the idiosyncratic enzyme active-site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aq. soln. Whereas structural inspection of systems has often attributed perturbed pKa values to dominant contributions from placement near charged groups or within hydrophobic pockets, Tyr57 of a Pseudomonas putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semisynthesis, 13C NMR spectroscopy, absorbance spectroscopy, and X-ray crystallog., was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of pos. charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from the introduction of nearby anionic groups, arises from accompanying active-site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water mol. that is part of a stabilizing hydrogen-bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen-bond donors, two Tyr side chains, and a water mol. positioned by other side chains and by a water-mediated hydrogen-bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural anal. can greatly facilitate our understanding of enzyme active-site energetics. The extensive data set provided may also be a valuable resource for those wishing to extensively test computational approaches for detg. enzymic pKa values and energetic effects.
- 307Hett, E. C.; Xu, H.; Geoghegan, K. F.; Gopalsamy, A.; Kyne, R. E.; Menard, C. A.; Narayanan, A.; Parikh, M. D.; Liu, S.; Roberts, L.; Robinson, R. P.; Tones, M. A.; Jones, L. H. Rational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride Probes. ACS Chem. Biol. 2015, 10 (4), 1094– 1098, DOI: 10.1021/cb5009475[ACS Full Text], [CAS], Google Scholar307https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmt1Kqug%253D%253D&md5=4af0b5188be76f67d43f307287571fdfRational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride ProbesHett, Erik C.; Xu, Hua; Geoghegan, Kieran F.; Gopalsamy, Ariamala; Kyne, Robert E.; Menard, Carol A.; Narayanan, Arjun; Parikh, Mihir D.; Liu, Shenping; Roberts, Lee; Robinson, Ralph P.; Tones, Michael A.; Jones, Lyn H.ACS Chemical Biology (2015), 10 (4), 1094-1098CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)This work describes the first rational targeting of tyrosine residues in a protein binding site by small-mol. covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based mol. design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition expts. with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that were used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chem. biol. toolkit by these probes uses in target validation and mol. pharmacol.
- 308Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation. J. Am. Chem. Soc. 2004, 126 (49), 15942– 15943, DOI: 10.1021/ja0439017[ACS Full Text], [CAS], Google Scholar308https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXpvFWjur8%253D&md5=d9cb096caf0ad9046b42c8e6bdb7145aA Three-Component Mannich-Type Reaction for Selective Tyrosine BioconjugationJoshi, Neel S.; Whitaker, Leanna R.; Francis, Matthew B.Journal of the American Chemical Society (2004), 126 (49), 15942-15943CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A new selective bioconjugation reaction is described for the modification of tyrosine residues on protein substrates. The reaction uses imines formed in situ from aldehydes and electron-rich anilines to modify phenolic side chains through a Mannich-type electrophilic arom. substitution pathway. The reaction takes place under mild pH and temp. conditions and can modify protein substrates at concns. as low as 20 μM. Using an efficient fluorescence-based assay, we demonstrated the reaction using a no. of aldehydes and protein targets. Importantly, proteins lacking surface-accessible tyrosines remained unmodified. It was also demonstrated that enzymic activity is preserved under the mild reaction conditions. This strategy represents one of the first carbon-carbon bond-forming reactions for protein modification and provides an important complement to more commonly used lysine- and cysteine-based methods.
- 309Jones, M. W.; Mantovani, G.; Blindauer, C. A.; Ryan, S. M.; Wang, X.; Brayden, D. J.; Haddleton, D. M. Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium Salts. J. Am. Chem. Soc. 2012, 134 (17), 7406– 7413, DOI: 10.1021/ja211855q[ACS Full Text], [CAS], Google Scholar309https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xltl2gtLw%253D&md5=fb9846543b44d6852e9fb52ca0b8c72fDirect Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium SaltsJones, Mathew W.; Mantovani, Giuseppe; Blindauer, Claudia A.; Ryan, Sinead M.; Wang, Xuexuan; Brayden, David J.; Haddleton, David M.Journal of the American Chemical Society (2012), 134 (17), 7406-7413CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Direct polymer conjugation at peptide tyrosine residues is described. In this study Tyr residues of both leucine enkephalin and salmon calcitonin (sCT) were targeted using appropriate diazonium salt-terminated linear monomethoxy poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacrylate prepd. by atom transfer radical polymn. Judicious choice of the reaction conditions-pH, stoichiometry, and chem. structure of diazonium salt-led to a high degree of site-specificity in the conjugation reaction, even in the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed that conjugation of mPEG2000 to sCT did not affect the peptide's ability to increase intracellular cAMP induced in T47D human breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca2+] plasma levels by mPEG2000-sCT conjugate in rat animal models.
- 310Ban, H.; Gavrilyuk, J.; Barbas, C. F. Tyrosine Bioconjugation through Aqueous Ene-Type Reactions: A Click-Like Reaction for Tyrosine. J. Am. Chem. Soc. 2010, 132 (5), 1523– 1525, DOI: 10.1021/ja909062q[ACS Full Text], [CAS], Google Scholar310https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkt1Wrug%253D%253D&md5=876ee34e149ceed4f70f7fd4efbb25baTyrosine bioconjugation through aqueous ene-type reactions: A click-like reaction for tyrosineBan, Hitoshi; Gavrilyuk, Julia; Barbas, Carlos F.Journal of the American Chemical Society (2010), 132 (5), 1523-1525CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A new and versatile class of cyclic diazodicarboxamides that reacts efficiently and selectively with phenols and the phenolic side chain of tyrosine through an ene-like reaction is reported. This mild aq. tyrosine ligation reaction works over a broad pH range and expands the repertoire of aq. chemistries available for small mol., peptide, and protein modification. The tyrosine ligation reactions are shown to be compatible with the labeling of native enzymes and antibodies in a buffered aq. soln. This reaction provides a novel synthetic approach to bispecific antibodies. The authors believe this reaction will find broad utility in peptide and protein chem. and in the chem. of phenol-contg. compds.
- 311Ban, H.; Nagano, M.; Gavrilyuk, J.; Hakamata, W.; Inokuma, T.; Barbas, C. F. Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction. Bioconjugate Chem. 2013, 24 (4), 520– 532, DOI: 10.1021/bc300665t[ACS Full Text], [CAS], Google Scholar311https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXkslWlsrY%253D&md5=7f548b32c1e16dd72adbe15b05bbf38cFacile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click ReactionBan, Hitoshi; Nagano, Masanobu; Gavrilyuk, Julia; Hakamata, Wataru; Inokuma, Tsubasa; Barbas, Carlos F., IIIBioconjugate Chemistry (2013), 24 (4), 520-532CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivs. in peptide and protein chem., we synthesized PTAD derivs. possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivs. and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective trifunctionalization of proteins is readily achieved. In particular cases, we noted that PTAD decompn. resulted in formation of a putative isocyanate byproduct that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addn. of a small amt. of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene glycol) chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen, whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody-drug conjugate. For this purpose, we synthesized a PTAD deriv. linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small mols. The tyrosine click linkage demonstrated stability to extremes of pH, temp., and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small mols., peptides, and proteins under mild aq. conditions over a broad pH range using a wide variety of biol. acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compns.
- 312Hatcher, J. M.; Wu, G.; Zeng, C.; Zhu, J.; Meng, F.; Patel, S.; Wang, W.; Ficarro, S. B.; Leggett, A. L.; Powell, C. E.; Marto, J. A.; Zhang, K.; Ngo, J. C. K.; Fu, X.-D.; Zhang, T.; Gray, N. S. SRPKIN-1: A Covalent SRPK1/2 Inhibitor That Potently Converts VEGF from Pro-Angiogenic to Anti-Angiogenic Isoform. Cell Chem. Biol. 2018, 25, 460– 470, DOI: 10.1016/j.chembiol.2018.01.013[Crossref], [PubMed], [CAS], Google Scholar312https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjt1Sgtrs%253D&md5=713e21a6f41894c0b0a0c876ef5ded79SRPKIN-1: A Covalent SRPK1/2 Inhibitor that Potently Converts VEGF from Pro-angiogenic to Anti-angiogenic IsoformHatcher, John M.; Wu, Guowei; Zeng, Chuyue; Zhu, Jie; Meng, Fan; Patel, Sherrina; Wang, Wenqiu; Ficarro, Scott B.; Leggett, Alan L.; Powell, Chelsea E.; Marto, Jarrod A.; Zhang, Kang; Ki Ngo, Jacky Chi; Fu, Xiang-Dong; Zhang, Tinghu; Gray, Nathanael S.Cell Chemical Biology (2018), 25 (4), 460-470.e6CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)The SRPK family of kinases regulates pre-mRNA splicing by phosphorylating serine/arginine (SR)-rich splicing factors, signals splicing control in response to extracellular stimuli, and contributes to tumorigenesis, suggesting that these splicing kinases are potential therapeutic targets. Here, we report the development of the first irreversible SRPK inhibitor, SRPKIN-1, which is also the first kinase inhibitor that forms a covalent bond with a tyrosine phenol group in the ATP-binding pocket. Kinome-wide profiling demonstrates its selectivity for SRPK1/2, and SRPKIN-1 attenuates SR protein phosphorylation at submicromolar concns. Vascular endothelial growth factor (VEGF) is a known target for SRPK-regulated splicing and, relative to the first-generation SRPK inhibitor SRPIN340 or small interfering RNA-mediated SRPK knockdown, SRPKIN-1 is more potent in converting the pro-angiogenic VEGF-A165a to the anti-angiogenic VEGF-A165b isoform and in blocking laser-induced neovascularization in a murine retinal model. These findings encourage further development of SRPK inhibitors for treatment of age-related macular degeneration.
- 313Patricelli, M. P.; Nomanbhoy, T. K.; Wu, J.; Brown, H.; Zhou, D.; Zhang, J.; Jagannathan, S.; Aban, A.; Okerberg, E.; Herring, C.; Nordin, B.; Weissig, H.; Yang, Q.; Lee, J.-D.; Gray, N. S.; Kozarich, J. W. In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases. Chem. Biol. 2011, 18 (6), 699– 710, DOI: 10.1016/j.chembiol.2011.04.011[Crossref], [PubMed], [CAS], Google Scholar313https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXotVejtbo%253D&md5=dda091b895850c95131f80c3aba9e8c7In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native KinasesPatricelli, Matthew P.; Nomanbhoy, Tyzoon K.; Wu, Jiangyue; Brown, Heidi; Zhou, David; Zhang, Jianming; Jagannathan, Subadhra; Aban, Arwin; Okerberg, Eric; Herring, Chris; Nordin, Brian; Weissig, Helge; Yang, Qingkai; Lee, Jiing-Dwan; Gray, Nathanael S.; Kozarich, John W.Chemistry & Biology (Cambridge, MA, United States) (2011), 18 (6), 699-710CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Summary: Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biol. targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Addnl., Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.
- 314Sakamoto, H.; Tsukaguchi, T.; Hiroshima, S.; Kodama, T.; Kobayashi, T.; Fukami, T. A.; Oikawa, N.; Tsukuda, T.; Ishii, N.; Aoki, Y. CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. Cancer Cell 2011, 19 (5), 679– 690, DOI: 10.1016/j.ccr.2011.04.004[Crossref], [PubMed], [CAS], Google Scholar314https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtFGlsL8%253D&md5=cae32f3a3647926ba835293b68e776f8CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper MutantSakamoto, Hiroshi; Tsukaguchi, Toshiyuki; Hiroshima, Sayuri; Kodama, Tatsushi; Kobayashi, Takamitsu; Fukami, Takaaki A.; Oikawa, Nobuhiro; Tsukuda, Takuo; Ishii, Nobuya; Aoki, YukoCancer Cell (2011), 19 (5), 679-690CODEN: CCAECI; ISSN:1535-6108. (Cell Press)Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers, following gene alterations such as chromosomal translocation, amplification, or point mutation. Here, the authors identified CH5424802, a potent, selective, and orally available ALK inhibitor with a unique chem. scaffold, showing preferential antitumor activity against cancers with gene alterations of ALK, such as non-small cell lung cancer (NSCLC) cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion in vitro and in vivo. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven cell growth. The authors' results support the potential for clin. evaluation of CH5424802 for the treatment of patients with ALK-driven tumors.
- 315Choi, E. J.; Jung, D.; Kim, J.-S.; Lee, Y.; Kim, B. M. Chemoselective Tyrosine Bioconjugation through Sulfate Click Reaction. Chem. - Eur. J. 2018, 24 (43), 10948– 10952, DOI: 10.1002/chem.201802380[Crossref], [PubMed], [CAS], Google Scholar315https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlCmt7vE&md5=f0bcca396c1439a2501cc04c98c9dde2Chemoselective Tyrosine Bioconjugation through Sulfate Click ReactionChoi, Eun Joung; Jung, Dongwook; Kim, Jong-Seo; Lee, Yan; Kim, B. MoonChemistry - A European Journal (2018), 24 (43), 10948-10952CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)A novel and selective tyrosine functionalization strategy through SuFEx (sulfur fluoride exchange) chem. is presented. In this approach, free tyrosine (Tyr) reacts selectively with aryl fluorosulfate in the presence of various nucleophilic amino acid residues in bio-tolerable conditions. Chemoselectivity of this unique SuFEx reaction was confirmed in amino acid, peptide, and protein conjugations. The functions of peptides and proteins were well-preserved as demonstrated from the Tyr-specific modification of cell-penetrating peptide and erythropoietin. This method is well-suited for residue-specific modification of native proteins, and thus would expand the versatility of bio-conjugation in protein chem.
- 316Fadeyi, O. O.; Hoth, L. R.; Choi, C.; Feng, X.; Gopalsamy, A.; Hett, E. C.; Kyne, R. E.; Robinson, R. P.; Jones, L. H. Covalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine Residue. ACS Chem. Biol. 2017, 12 (8), 2015– 2020, DOI: 10.1021/acschembio.7b00403[ACS Full Text], [CAS], Google Scholar316https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFyisLfI&md5=d9b49c1f7d95cba028a1eb7a9f8e776aCovalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine ResidueFadeyi, Olugbeminiyi O.; Hoth, Lise R.; Choi, Chulho; Feng, Xidong; Gopalsamy, Ariamala; Hett, Erik C.; Kyne, Robert E., Jr.; Robinson, Ralph P.; Jones, Lyn H.ACS Chemical Biology (2017), 12 (8), 2015-2020CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Irreversible enzyme inhibitors and covalent chem. biol. probes often utilize the reaction of a protein cysteine residue with an appropriately positioned electrophile (e.g., acrylamide) on the ligand template. However, cysteine residues are not always available for site-specific protein labeling, and therefore new approaches are needed to expand the toolkit of appropriate electrophiles ("warheads") that target alternative amino acids. We previously described the rational targeting of tyrosine residues in the active site of a protein (the mRNA decapping scavenger enzyme, DcpS) using inhibitors armed with a sulfonyl fluoride electrophile. These inhibitors subsequently enabled the development of clickable probe technol. to measure drug-target occupancy in live cells. Here we describe a fluorosulfate-contg. inhibitor (aryl fluorosulfate probe (FS-p1)) with excellent chem. and metabolic stability that reacts selectively with a noncatalytic serine residue in the same active site of DcpS as confirmed by peptide mapping expts. Our results suggest that noncatalytic serine targeting using fluorosulfate electrophilic warheads could be a suitable strategy for the development of covalent inhibitor drugs and chem. probes.
- 317Crawford, L. A.; Weerapana, E. A Tyrosine-Reactive Irreversible Inhibitor for Glutathione S-Transferase Pi (GSTP1). Mol. BioSyst. 2016, 12 (6), 1768– 1771, DOI: 10.1039/C6MB00250A[Crossref], [PubMed], [CAS], Google Scholar317https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsF2ntb8%253D&md5=f30c8260bb5e863fe8f0d70fb3aef15dA tyrosine-reactive irreversible inhibitor for glutathione S-transferase Pi (GSTP1)Crawford, L. A.; Weerapana, E.Molecular BioSystems (2016), 12 (6), 1768-1771CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)Glutathione S-transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, the authors report LAS17, a dichlorotriazine-contg. compd. that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition.
- 318Gehringer, M.; Forster, M.; Pfaffenrot, E.; Bauer, S. M.; Laufer, S. A. Novel Hinge-Binding Motifs for Janus Kinase 3 Inhibitors: A Comprehensive Structure–Activity Relationship Study on Tofacitinib Bioisosteres. ChemMedChem 2014, 9 (11), 2516– 2527, DOI: 10.1002/cmdc.201402252[Crossref], [PubMed], [CAS], Google Scholar318https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlOksrnE&md5=f1ca6c4bf272b41b5204b8b82ea5836eNovel hinge-binding motifs for janus kinase 3 inhibitors: a comprehensive structure-activity relationship study on tofacitinib bioisosteresGehringer, Matthias; Forster, Michael; Pfaffenrot, Ellen; Bauer, Silke M.; Laufer, Stefan A.ChemMedChem (2014), 9 (11), 2516-2527CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The Janus kinases (JAKs) are a family of cytosolic tyrosine kinases crucially involved in cytokine signaling. JAKs have been demonstrated to be valid targets in the treatment of inflammatory and myeloproliferative disorders, and two inhibitors, tofacitinib and ruxolitinib, recently received their marketing authorization. Despite this success, selectivity within the JAK family remains a major issue. Both approved compds. share a common 7H-pyrrolo[2,3-d]pyrimidine hinge binding motif, and little is known about modifications tolerated at this heterocyclic core. In the current study, a library of tofacitinib bioisosteres was prepd. and tested against JAK3. The compds. possessed the tofacitinib piperidinyl side chain, whereas the hinge binding motif was replaced by a variety of heterocycles mimicking its pharmacophore. In view of the promising expectations obtained from mol. modeling, most of the compds. proved to be poorly active. However, strategies for restoring activity within this series of novel chemotypes were discovered and crucial structure-activity relationships were deduced. The compds. presented may serve as starting point for developing novel JAK inhibitors and as a valuable training set for in silico models.
- 319Gu, C.; Shannon, D. A.; Colby, T.; Wang, Z.; Shabab, M.; Kumari, S.; Villamor, J. G.; McLaughlin, C. J.; Weerapana, E.; Kaiser, M.; Cravatt, B. F.; van der Hoorn, R. A. L. Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases. Chem. Biol. 2013, 20 (4), 541– 548, DOI: 10.1016/j.chembiol.2013.01.016[Crossref], [PubMed], [CAS], Google Scholar319https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXmt1Clsbo%253D&md5=71d3ce60eed76d8a361fc0057d2102e1Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione TransferasesGu, Christian; Shannon, D. Alexander; Colby, Tom; Wang, Zheming; Shabab, Mohammed; Kumari, Selva; Villamor, Joji Grace; McLaughlin, Christopher J.; Weerapana, Eranthie; Kaiser, Markus; Cravatt, Benjamin F.; van der Hoorn, Renier A. L.Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (4), 541-548CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)Chem. probes have great potential for identifying functional residues in proteins in crude proteomes. Here we studied labeling sites of chem. probes based on sulfonyl fluorides (SFs) on plant and animal proteomes. Besides serine proteases and many other proteins, SF-based probes label Tyr residues in glutathione transferases (GSTs). The labeled GSTs represent four different GST classes that share less than 30% sequence identity. The targeted Tyr residues are located at similar positions in the promiscuous substrate binding site and are essential for GST function. The high selectivity of SF-based probes for functional Tyr residues in GSTs illustrates how these probes can be used for functional studies of GSTs and other proteins in crude proteomes.
- 320Ekici, Ö. D.; Paetzel, M.; Dalbey, R. E. Unconventional Serine Proteases: Variations on the Catalytic Ser/His/Asp Triad Configuration. Protein Sci. 2008, 17 (12), 2023– 2037, DOI: 10.1110/ps.035436.108[Crossref], [PubMed], [CAS], Google Scholar320https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVKgt7rP&md5=ad4e54f8dd98af7f51999678806d40acUnconventional serine proteases: variations on the catalytic Ser/His/Asp triad configurationEkici, Ozlem Dogan; Paetzel, Mark; Dalbey, Ross E.Protein Science (2008), 17 (12), 2023-2037CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)A review. Serine proteases comprise nearly 1/3 of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called "classical" catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of "nonclassical" serine proteases where variations to the catalytic triad are obsd. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine proteases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. Here, the authors discuss the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.
- 321Long, J. Z.; Cravatt, B. F. The Metabolic Serine Hydrolases and Their Functions in Mammalian Physiology and Disease. Chem. Rev. 2011, 111 (10), 6022– 6063, DOI: 10.1021/cr200075y[ACS Full Text], [CAS], Google Scholar321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvFagtb8%253D&md5=c978f77bd911d5877df4ba97fc09e4b3The metabolic serine hydrolases and their functions in mammalian physiology and diseaseLong, Jonathan Z.; Cravatt, Benjamin F.Chemical Reviews (Washington, DC, United States) (2011), 111 (10), 6022-6063CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Serine hydrolases (SHs) consist of >200 enzymes in humans which are characterized by the presence of an active site Ser residue that is used for the hydrolysis of substrates. The membership of this enzyme class is nearly equally split between serine proteases (trypsin/chymotrypsin/subtilisin enzymes) and "metabolic" SHs that cleave ester, amide, or thioester bonds in small mols., peptides, or proteins. The focus of this review is on the metabolic SHs. Here, the authors attempt to provide a comprehensive summary that captures the state of knowledge about metabolic SHs in their entirety, including those enzymes that remain mostly or completely uncharacterized. Particular emphasis is placed on relating the biochem. and enzymol. of individual SHs to the physiol. substrates and products they regulate in living systems, and how SHs, through the regulation of specific metabolic pathways impact health and disease.
- 322Lei, J.; Zhou, Y.; Xie, D.; Zhang, Y. Mechanistic Insights into a Classic Wonder Drug—Aspirin. J. Am. Chem. Soc. 2015, 137 (1), 70– 73, DOI: 10.1021/ja5112964[ACS Full Text], [CAS], Google Scholar322https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFOgsLzI&md5=ec731b98115bd7b896b23d97087b24c3Mechanistic Insights into a Classic Wonder Drug-AspirinLei, Jinping; Zhou, Yanzi; Xie, Daiqian; Zhang, YingkaiJournal of the American Chemical Society (2015), 137 (1), 70-73CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Aspirin, one of the oldest and most common anti-inflammatory agents, has recently been shown to reduce cancer risks. The principal pharmacol. effects of aspirin are known to arise from its covalent modification of cyclooxygenase-2 (COX-2) through acetylation of Ser530, but the detailed mechanism of its biochem. action and specificity remains to be elucidated. In this work, we have filled this gap by employing a state-of-the-art computational approach, Born-Oppenheimer mol. dynamics simulations with ab initio quantum mech./mol. mech. potential and umbrella sampling. Our studies have characterized a substrate-assisted inhibition mechanism for aspirin acetylating COX: it proceeds in two successive stages with a metastable tetrahedral intermediate, in which the carboxyl group of aspirin serves as the general base. The computational results confirmed that aspirin would be 10-100 times more potent against COX-1 than against COX-2, and revealed that this inhibition specificity between the two COX isoforms can be attributed mainly to the difference in kinetics rate of the covalent inhibition reaction, not the aspirin-binding step. The structural origin of this differential inhibition of the COX enzymes by aspirin has also been elucidated.
- 323Leney, A. C.; Heck, A. J. R. Native Mass Spectrometry: What Is in the Name?. J. Am. Soc. Mass Spectrom. 2017, 28 (1), 5– 13, DOI: 10.1007/s13361-016-1545-3[ACS Full Text], [CAS], Google Scholar323https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFylsLzF&md5=09d56e0a888ad31a6f655c4e1d840775Native Mass Spectrometry: What is in the Name?Leney, Aneika C.; Heck, Albert J. R.Journal of the American Society for Mass Spectrometry (2017), 28 (1), 5-13CODEN: JAMSEF; ISSN:1044-0305. (Springer)A review. Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomol. mass spectrometry and proteomics. Advances in sample prepn. and mass analyzers have enabled researchers to ext. much more information from biol. samples than just the mol. wt. In particular, relevant for structural biol., noncovalent protein-protein and protein-ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomol. mass spectrometry, nowadays often referred to as "native MS," has come to maturation over the last two decades, with dozens of labs. using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure-function relations. In this perspective, the authors describe the origins of and (re)define the term native MS, portraying in detail what the authors meant by "native MS," when the term was coined and also describing what it does (according to the authors) not entail. Addnl., the authors describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technol. has in solving complex biol. questions.
- 324Li, Z.; Qian, L.; Li, L.; Bernhammer, J. C.; Huynh, H. V.; Lee, J.-S.; Yao, S. Q. Tetrazole Photoclick Chemistry: Reinvestigating Its Suitability as a Bioorthogonal Reaction and Potential Applications. Angew. Chem., Int. Ed. 2016, 55 (6), 2002– 2006, DOI: 10.1002/anie.201508104[Crossref], [CAS], Google Scholar324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFKisbjM&md5=6438d371472471f5e43159dc60712774Tetrazole Photoclick Chemistry: Reinvestigating Its Suitability as a Bioorthogonal Reaction and Potential ApplicationsLi, Zhengqiu; Qian, Linghui; Li, Lin; Bernhammer, Jan C.; Huynh, Han Vinh; Lee, Jun-Seok; Yao, Shao Q.Angewandte Chemie, International Edition (2016), 55 (6), 2002-2006CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The bioorthogonality of tetrazole photoclick chem. has been reassessed. Upon photolysis of a tetrazole, the highly reactive nitrile imine formed undergoes rapid nucleophilic reaction with a variety of nucleophiles present in a biol. system, along with the expected cycloaddn. with alkenes. The alternative use of the tetrazole photoclick reaction was thus explored: tetrazoles were incorporated into Bodipy and Acedan dyes, providing novel photo-crosslinkers with one- and two-photon fluorescence Turn-ON properties that may be developed into protein-detecting biosensors. Further introduction of these photo-activatable, fluorogenic moieties into staurosporine resulted in the corresponding probes capable of photoinduced, no-wash imaging of endogenous kinase activities in live mammalian cells.
- 325Zhao, S.; Dai, J.; Hu, M.; Liu, C.; Meng, R.; Liu, X.; Wang, C.; Luo, T. Photo-Induced Coupling Reactions of Tetrazoles with Carboxylic Acids in Aqueous Solution: Application in Protein Labelling. Chem. Commun. 2016, 52 (25), 4702– 4705, DOI: 10.1039/C5CC10445A[Crossref], [PubMed], [CAS], Google Scholar325https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs1ejs78%253D&md5=09c69da8265c035f3dcd8000d298c0b8Photo-induced coupling reactions of tetrazoles with carboxylic acids in aqueous solution: application in protein labelingZhao, Shan; Dai, Jianye; Hu, Mo; Liu, Chang; Meng, Rong; Liu, Xiaoyun; Wang, Chu; Luo, TuopingChemical Communications (Cambridge, United Kingdom) (2016), 52 (25), 4702-4705CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)The photo-induced reactions of diaryltetrazoles with carboxylic acids in aq. soln. were studied. Besides measuring the apparent second-order rate const. and evaluating the functional group compatibility of these reactions, the authors further incorporated the tetrazoles into SAHA, leading to a new active-site-directed probe for labeling HDACs in both cell lysates and living cells.
- 326Mix, K. A.; Raines, R. T. Optimized Diazo Scaffold for Protein Esterification. Org. Lett. 2015, 17 (10), 2358– 2361, DOI: 10.1021/acs.orglett.5b00840[ACS Full Text], [CAS], Google Scholar326https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXnvV2ktrc%253D&md5=5737bf6bf6f0a391613597bd2e4d906bOptimized Diazo Scaffold for Protein EsterificationMix, Kalie A.; Raines, Ronald T.Organic Letters (2015), 17 (10), 2358-2361CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)The O-alkylation of carboxylic acids with diazo compds. provides a means to esterify carboxylic acids in aq. soln. A Hammett anal. of the reactivity of diazo compds. derived from phenylglycinamide revealed that the (p-methylphenyl)glycinamide scaffold has an esp. high reaction rate and ester/alc. product ratio and esterifies protein carboxyl groups more efficiently than any known reagent.
- 327Ban, H. S.; Usui, T.; Nabeyama, W.; Morita, H.; Fukuzawa, K.; Nakamura, H. Discovery of Boron-Conjugated 4-Anilinoquinazoline as a Prolonged Inhibitor of EGFR Tyrosine Kinase. Org. Biomol. Chem. 2009, 7 (21), 4415– 4427, DOI: 10.1039/b909504g[Crossref], [PubMed], [CAS], Google Scholar327https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1Gksb%252FN&md5=b61ee61865bdff8e4706485b1f030554Discovery of boron-conjugated 4-anilinoquinazoline as a prolonged inhibitor of EGFR tyrosine kinaseBan, Hyun Seung; Usui, Taikou; Nabeyama, Wataru; Morita, Hidetoshi; Fukuzawa, Kaori; Nakamura, HiroyukiOrganic & Biomolecular Chemistry (2009), 7 (21), 4415-4427CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Boron-conjugated 4-anilinoquinazolines were designed and synthesized as inhibitors of EGFR tyrosine kinase with possible covalent bond interactions between the boron atom and the nucleophilic groups of the EGFR kinase domain. Among the compds. synthesized, compds. I and II (X = CH:CH, 1,4-phenylene) reduced the EGF-mediated phosphorylation of EGFR tyrosine kinase and its downstream kinases including ERK and Akt in A431 cells. The cell growth was inhibited by these compds. through arrest of G1 cell cycle, which induced apoptosis. A time-dependent in vitro preincubation assay demonstrated the irreversible inhibition of compd. II [X = 1,4-phenylene; (III)] against EGFR tyrosine kinase. Quantum mech. docking simulation revealed that the boronic acid moiety of compd. III formed a covalent B-O bond with Asp800 in addn. to hydrogen bonds with Asp800 and Cys797, which may cause the prolonged inhibition of compd. III toward EGFR tyrosine kinase.
- 328Woodward, R. B.; Olofson, R. A.; Mayer, H. A New Synthesis of Peptides. J. Am. Chem. Soc. 1961, 83 (4), 1010– 1012, DOI: 10.1021/ja01465a072[ACS Full Text], [CAS], Google Scholar328https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3MXnsFyhsQ%253D%253D&md5=52b956257b3ee4dff7701172de0a9d4cA new synthesis of peptidesWoodward, R. B.; Olofson, R. A.; Mayer, HansJournal of the American Chemical Society (1961), 83 (), 1010-12CODEN: JACSAT; ISSN:0002-7863.N-Ethyl-5-phenylisoxazolium-3'-sulfonate (I) reacted readily with carboxylates to serve as the carboxyl-activating step in a new simple synthesis of peptides. A protected amino acid or peptide (1 mole) dissolved in MeCN or MeNO2 contg. 1 mole Et3N was added to 1 mole I in the same solvent and stirred at 0° until soln. was complete (activation step). The amino acid ester-HCl or peptide ester-HCl (1 mole) and an equiv. amt. Et3N were added and stirred overnight at room temp. The solvent was removed and the mixt. worked up. Several examples were carried out with activation in MeCN at 0° for approx. 1 hr. (peptide, yield, m.p. given): L-PhCH2CH(NHZ)CONHCH2CO2Et, 93, 109-10°; o-C6H4(CO)2NCH2CONHCH2CO2Et, 88, 193.5-4.5°; L-ZHN(CH2)4CH(NHZ)CHCONHCH2CO2Et, 95, 90.5-2.5°; L-PhCH2CH(NHZ)CONHCH(CH2CHMe2)CO2Me-L, 90, 106-7°; L-ZHNCH(CH2CONH2)CONHCH2CO2Et (II), 79, 186-7°; L-PhCH2CH(HNOCCH2NHZ)CONHCH2CO2Et, 90, 116.5-7.5°; L-(4-HOC6H4)CH2CH(HNOCCH2NHOCCH2NHZ)CO2Me, 84, 159.5-61.5°; ZHNCH2CO(NHCH2CO)2OEt, 91, 167-8°; L-ZHNCH(CH2CH2SMe)CO(HNCH2CO)2OEt, 86, 131.5-3°; L-ZN.CH2.CH2.CH(OH).CHCO(HNCH2CO)2OEt, 80, 145-6°; and with activation in MeNO2 at room temp. for 7 min. (same data given): II, 80, 185.5-7.0°; L-ZHNCH(CH2CONH2)CONHCH(CH2CHMe2)CO2Me-L, 76, 176.5-8.0°; L-ZHNCH(CH2CH2CONH2)CONHCH(CHMe2)CO2Me-L, 77, 172.5-3.0°; L-ZHNCH(CH2CH2CONH2)CONHCH(CH2C6H4OH - 4)CO2Me-L, 75, 198-9°; ZHNCH2CO(HNCH2CO)2OEt, 90, 167-8°; DL-PhCH2CH(HNOCCH2NHZ)CONHCH2CO2Et, 89, 132-3°.
- 329Martín-Gago, P.; Fansa, E. K.; Winzker, M.; Murarka, S.; Janning, P.; Schultz-Fademrecht, C.; Baumann, M.; Wittinghofer, A.; Waldmann, H. Covalent Protein Labeling at Glutamic Acids. Cell Chem. Biol. 2017, 24, 589– 597, DOI: 10.1016/j.chembiol.2017.03.015[Crossref], [PubMed], [CAS], Google Scholar329https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmsVWiur4%253D&md5=82057dbd875a929697a36ff86c532160Covalent Protein Labeling at Glutamic AcidsMartin-Gago, Pablo; Fansa, Eyad K.; Winzker, Michael; Murarka, Sandip; Janning, Petra; Schultz-Fademrecht, Carsten; Baumann, Matthias; Wittinghofer, Alfred; Waldmann, HerbertCell Chemical Biology (2017), 24 (5), 589-597.e5CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Covalent labeling of amino acids in proteins by reactive small mols., in particular at cysteine SH and lysine NH groups, is a powerful approach to identify and characterize proteins and their functions. However, for the less-reactive carboxylic acids present in Asp and Glu, hardly any methodol. is available. Employing the lipoprotein binding chaperone PDE6δ as an example, we demonstrate that incorporation of isoxazolium salts that resemble the structure and reactivity of Woodward's reagent K into protein ligands provides a novel method for selective covalent targeting of binding site carboxylic acids in whole proteomes. Covalent adduct formation occurs via rapid formation of enol esters and the covalent bond is stable even in the presence of strong nucleophiles. This new method promises to open up hitherto unexplored opportunities for chem. biol. research.
- 330Martín-Gago, P.; Fansa, E. K.; Klein, C. H.; Murarka, S.; Janning, P.; Schürmann, M.; Metz, M.; Ismail, S.; Schultz-Fademrecht, C.; Baumann, M.; Bastiaens, P. I. H.; Wittinghofer, A.; Waldmann, H. A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity That Prevents Efficient Inhibitor Release by Arl2. Angew. Chem., Int. Ed. 2017, 56 (9), 2423– 2428, DOI: 10.1002/anie.201610957[Crossref], [CAS], Google Scholar330https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsValt7k%253D&md5=2b66846ac0d9806f58b428d83819a7c7A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2Martin-Gago, Pablo; Fansa, Eyad K.; Klein, Christian H.; Murarka, Sandip; Janning, Petra; Schuermann, Marc; Metz, Malte; Ismail, Shehab; Schultz-Fademrecht, Carsten; Baumann, Matthias; Bastiaens, Philippe I. H.; Wittinghofer, Alfred; Waldmann, HerbertAngewandte Chemie, International Edition (2017), 56 (9), 2423-2428CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Small-mol. inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro (KD<10 nM), interference with Ras signaling and growth inhibition require 5-20 μm compd. concns. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compds. selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas.
- 331Tsukiji, S.; Hamachi, I. Ligand-Directed Tosyl Chemistry for in Situ Native Protein Labeling and Engineering in Living Systems: From Basic Properties to Applications. Curr. Opin. Chem. Biol. 2014, 21, 136– 143, DOI: 10.1016/j.cbpa.2014.07.012[Crossref], [PubMed], [CAS], Google Scholar331https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVOhs7nK&md5=0e1580b982702ef2074672524209f5e2Ligand-directed tosyl chemistry for in situ native protein labeling and engineering in living systems: from basic properties to applicationsTsukiji, Shinya; Hamachi, ItaruCurrent Opinion in Chemical Biology (2014), 21 (), 136-143CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. The ability to introduce any chem. probe to any endogenous target protein in its native environment, that is in cells and in vivo, is anticipated to provide various new exciting tools for biol. and biomedical research. Although still at the prototype stage, the ligand-directed tosyl (LDT) chem. is a novel type of affinity labeling technique that we developed for such a dream. This chem. allows for modifying native proteins by various chem. probes with high specificity in various biol. settings ranging from in vitro (in test tubes) to in living cells and in vivo. Since the first report, the list of proteins that are successfully labeled by the LDT chem. has been increasing. A growing no. of studies have demonstrated its utility to create semisynthetic proteins directly in cellular contexts. The in situ generated semisynthetic proteins are applicable for various types of anal. and imaging of intracellular biol. processes. In this review, we summarize the basic properties of the LDT chem. and its applications toward in situ engineering and anal. of native proteins in living systems. Current limitations and future challenges of this area are also described.
- 332Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundbäck, T.; Nordlund, P.; Molina, D. M. The Cellular Thermal Shift Assay for Evaluating Drug Target Interactions in Cells. Nat. Protoc. 2014, 9 (9), 2100– 2122, DOI: 10.1038/nprot.2014.138[Crossref], [PubMed], [CAS], Google Scholar332https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht12msrfJ&md5=e7852e1a306bca1698a8e8463ae566b1The cellular thermal shift assay for evaluating drug target interactions in cellsJafari, Rozbeh; Almqvist, Helena; Axelsson, Hanna; Ignatushchenko, Marina; Lundbaeck, Thomas; Nordlund, Paer; Molina, Daniel MartinezNature Protocols (2014), 9 (9), 2100-2122CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Thermal shift assays are used to study thermal stabilization of proteins upon ligand binding. Such assays have been used extensively on purified proteins in the drug discovery industry and in academia to detect interactions. Recently, we published a proof-of-principle study describing the implementation of thermal shift assays in a cellular format, which we call the cellular thermal shift assay (CETSA). The method allows studies of target engagement of drug candidates in a cellular context, herein exemplified with exptl. data on the human kinases p38α and ERK1/2. The assay involves treatment of cells with a compd. of interest, heating to denature and ppt. proteins, cell lysis, and the sepn. of cell debris and aggregates from the sol. protein fraction. Whereas unbound proteins denature and ppt. at elevated temps., ligand-bound proteins remain in soln. We describe two procedures for detecting the stabilized protein in the sol. fraction of the samples. One approach involves sample workup and detection using quant. western blotting, whereas the second is performed directly in soln. and relies on the induced proximity of two target-directed antibodies upon binding to sol. protein. The latter protocol has been optimized to allow an increased throughput, as potential applications require large nos. of samples. Both approaches can be completed in a day.
- 333Franken, H.; Mathieson, T.; Childs, D.; Sweetman, G. M. A.; Werner, T.; Tögel, I.; Doce, C.; Gade, S.; Bantscheff, M.; Drewes, G.; Reinhard, F. B. M.; Huber, W.; Savitski, M. M. Thermal Proteome Profiling for Unbiased Identification of Direct and Indirect Drug Targets Using Multiplexed Quantitative Mass Spectrometry. Nat. Protoc. 2015, 10 (10), 1567– 1593, DOI: 10.1038/nprot.2015.101[Crossref], [PubMed], [CAS], Google Scholar333https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVyrtbzN&md5=d9c225616a35fc5dcc97db234452d7e1Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometryFranken, Holger; Mathieson, Toby; Childs, Dorothee; Sweetman, Gavain M. A.; Werner, Thilo; Toegel, Ina; Doce, Carola; Gade, Stephan; Bantscheff, Marcus; Drewes, Gerard; Reinhard, Friedrich B. M.; Huber, Wolfgang; Savitski, Mikhail M.Nature Protocols (2015), 10 (10), 1567-1593CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)The direct detection of drug-protein interactions in living cells is a major challenge in drug discovery research. Recently, the authors introduced an approach termed thermal proteome profiling (TPP), which enables the monitoring of changes in protein thermal stability across the proteome using quant. mass spectrometry. The authors detd. the intracellular thermal profiles for up to 7000 proteins, and by comparing profiles derived from cultured mammalian cells in the presence or absence of a drug it was possible to identify direct and indirect targets of drugs in living cells in an unbiased manner. Here the authors demonstrate the complete workflow using the histone deacetylase inhibitor panobinostat. The key to this approach is the use of isobaric tandem mass tag 10-plex (TMT10) reagents to label digested protein samples corresponding to each temp. point in the melting curve so that the samples can be analyzed by multiplexed quant. mass spectrometry. Important steps in the bioinformatic anal. include data normalization, melting curve fitting and statistical significance detn. of compd. concn.-dependent changes in protein stability. All anal. tools are made freely available as R and Python packages. The workflow can be completed in 2 wk.
- 334Komissarov, A. A.; Romanova, D. V.; Debabov, V. G. Complete Inactivation of Escherichia Coli Uridine Phosphorylase by Modification of Asp5 with Woodward’s Reagent K. J. Biol. Chem. 1995, 270 (17), 10050– 10055, DOI: 10.1074/jbc.270.17.10050[Crossref], [PubMed], [CAS], Google Scholar334https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXltlWns7w%253D&md5=5848107c614fd0e1adc180b05dc63e23Complete inactivation of Escherichia coli uridine phosphorylase by modification of Asp5 with Woodward's reagent KKomissarov, Andrey A.; Romanova, Darya V.; Debabov, Vladimir G.Journal of Biological Chemistry (1995), 270 (17), 10050-5CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site (Ki = 0.07 mM) with subsequent modification of a carboxyl (k2 = 1.2 min-1). Neither substrate alone protected uridine phosphorylase from inactivation. The presence of phosphate did not affect the Ki and k2 values. The addn. of uracil or uridine led to a significant increase of both Ki (to 2.5 or 2.1 mM, resp.) and k3 (to 6.1 or 4.8 min-1, resp.) values. Thus, WRK could react in accordance with slow (high affinity) and fast (low affinity) mechanisms. Combined addn. of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (Ser4-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys13). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp5 carboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Km values for phosphate and uridine were changed from 5.1 and 0.19 mM (or 7.3 and 0.14 mM according to Leer et al. (Leer, J. C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, resp. The hydroxamic enzyme had higher thermostability than the native enzyme. The results obtained demonstrated the importance of the carboxyl at position 5. The loss of activity after selective group replacement is due to impaired stabilization of the transition state rather than to a decline in substrate affinity or change of the active site structure.
- 335Qian, Y.; Schürmann, M.; Janning, P.; Hedberg, C.; Waldmann, H. Activity-Based Proteome Profiling Probes Based on Woodward’s Reagent K with Distinct Target Selectivity. Angew. Chem., Int. Ed. 2016, 55 (27), 7766– 7771, DOI: 10.1002/anie.201602666[Crossref], [CAS], Google Scholar335https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnsFWlt7k%253D&md5=2b544a0647a5c81b883e2be0791f04d9Activity-Based Proteome Profiling Probes Based on Woodward's Reagent K with Distinct Target SelectivityQian, Yong; Schuermann, Marc; Janning, Petra; Hedberg, Christian; Waldmann, HerbertAngewandte Chemie, International Edition (2016), 55 (27), 7766-7771CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Woodward's reagent K (WRK) is a reactive heterocyclic compd. that has been employed in protein chem. to covalently and unspecifically label proteins at nucleophilic amino acids, notably at histidine and cysteine. The authors have developed a panel of WRK-derived activity-based probes and show that surprisingly and unexpectedly, these probes are fairly selective for a few proteins in the human proteome. The WRK-derived probes show unique reactivity towards the catalytic N-terminal proline in the macrophage migration inhibitory factor (MIF) and can be used to label and, if equipped with a fluorophore, to image MIF activities in living cells.
- 336Harlow, K. W.; Switzer, R. L. Chemical Modification of Salmonella Typhimurium Phosphoribosylpyrophosphate Synthetase with 5′-(p-Fluorosulfonylbenzoyl)Adenosine. Identification of an Active Site Histidine. J. Biol. Chem. 1990, 265, 5487– 5493[PubMed], [CAS], Google Scholar336https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXitFejtLg%253D&md5=84e395a2c7b0d7ff965ba1659a791091Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidineHarlow, Kenneth W.; Switzer, Robert L.Journal of Biological Chemistry (1990), 265 (10), 5487-93CODEN: JBCHA3; ISSN:0021-9258.Liq. chromatog. procedures were developed for rapidly locating the site of reaction of chem. modification reagents with S. typhimurium 5-phosphoribosyl-α-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent, 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol FSBA/mol enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolyzates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence, Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was assocd. with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicated histidine-130 as the site of reaction with FSBA.
- 337Uchida, K.; Stadtman, E. R. Modification of Histidine Residues in Proteins by Reaction with 4-Hydroxynonenal. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (10), 4544– 4548, DOI: 10.1073/pnas.89.10.4544[Crossref], [PubMed], [CAS], Google Scholar337https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XksVeqtL4%253D&md5=e319c0a1200aa40784b00fde9bdd7cd6Modification of histidine residues in proteins by reaction with 4-hydroxynonenalUchida, Koji; Stadtman, E. R.Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (10), 4544-8CODEN: PNASA6; ISSN:0027-8424.Histidine residues in proteins are major targets for reaction with the lipid peroxidn. product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivs. upon subsequent treatment with NaB[3H]H4. Amino acid anal. of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stoichiometric formation of 3H-labeled amino acid hydrolyzates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the prodn. of two compds. Upon acid hydrolysis, both derivs. yielded stoichiometric amts. of histidine. However, after redn. with NaBH4, acid hydrolysis led to a mixt. of amino acid derivs. [presumably, isomeric forms of Nπ(Nτ)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addn. of the imidazole nitrogen atom of histidine to the α,β-unsatd. bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be detd.
- 338Yamaguchi, S.; Aldini, G.; Ito, S.; Morishita, N.; Shibata, T.; Vistoli, G.; Carini, M.; Uchida, K. Δ12-Prostaglandin J2 as a Product and Ligand of Human Serum Albumin: Formation of an Unusual Covalent Adduct at His146. J. Am. Chem. Soc. 2010, 132 (2), 824– 832, DOI: 10.1021/ja908878n[ACS Full Text], [CAS], Google Scholar338https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFKgu73J&md5=40501d528aa461e802e773930952b533Δ12-Prostaglandin J2 as a product and ligand of human serum albumin: Formation of an unusual covalent adduct at His146Yamaguchi, Satoru; Aldini, Giancarlo; Ito, Sohei; Morishita, Nozomi; Shibata, Takahiro; Vistoli, Giulio; Carini, Marina; Uchida, KojiJournal of the American Chemical Society (2010), 132 (2), 824-832CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Human serum albumin (HSA), the most abundant protein in plasma, has a very unique function, catalyzing the conversion of prostaglandin J2 (PGJ2), a dehydration product of PGD2, to yield Δ12-PGJ2. These PGD2 metabolites are actively transported into cells and accumulated in the nuclei, where they act as potent inducers of cell growth inhibition and cell differentiation, and exhibit their own unique spectrum of biol. effects. The facts that (i) arachidonic acid metabolites bind to human serum albumin (HSA) and the metab. of these mols. is altered as a result of binding, (ii) HSA catalyzes the transformation of PGJ2 into Δ12-PGJ2, and (iii) Δ12-PGJ2 is stable in serum suggest that HSA may bind and stabilize Δ12-PGJ2 in a specific manner. A mol. interaction anal. using surface plasmon resonance (Biacore) indeed suggested the presence of a specific Δ12-PGJ2-binding site in HSA. To investigate the mol. details of the binding of this PGD2 metabolite to albumin, we analyzed the cocrystal structure of the HSA-Δ12-PGJ2-myristate complex by X-ray crystallog. and found that two Δ12-PGJ2 mols. bind to a primary site in subdomain IB of the protein. The electron d. results suggested that one of the two Δ12-PGJ2 mols. that specifically bind to the site covalently interacted with a histidine residue (His146). Using nano-LC-MS/MS anal. of the HSA-Δ12-PGJ2 complex, the formation of an unusual Δ12-PGJ2-histidine adduct at His146 was confirmed. Thus, our crystallog. and mass spectrometric analyses of the HSA-Δ12-PGJ2 complex provided intriguing new insights into the mol. details of how this electrophilic ligand interacts with its primary producer and transporter.
- 339Yoshizawa, M.; Itoh, T.; Hori, T.; Kato, A.; Anami, Y.; Yoshimoto, N.; Yamamoto, K. Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands. J. Med. Chem. 2018, 61 (14), 6339– 6349, DOI: 10.1021/acs.jmedchem.8b00774[ACS Full Text], [CAS], Google Scholar339https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtF2htLzN&md5=5cc7c5b614f89aaa7582a0a25225e2e8Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic LigandsYoshizawa, Mami; Itoh, Toshimasa; Hori, Tatsuya; Kato, Akira; Anami, Yasuaki; Yoshimoto, Nobuko; Yamamoto, KeikoJournal of Medicinal Chemistry (2018), 61 (14), 6339-6349CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We designed and synthesized vitamin D analogs with an electrophile as covalent modifiers for the vitamin D receptor (VDR). Novel vitamin D analogs 1-4 have an electrophilic enone group at the side chain for conjugate addn. to His301 or His393 in the VDR. All compds. showed specific VDR-binding potency and agonistic activity. Covalent bond formations of 1-4 with the ligand-binding domain (LBD) of VDR were evaluated by electrospray ionization mass spectrometry. All compds. were shown to covalently bind to the VDR-LBD, and the abundance of VDR-LBD corresponding conjugate adducts of 1-4 increased with incubation time. Enone compds. 1 and 2 showed higher reactivity than the ene-ynone 3 and dienone 4 compds. Furthermore, we successfully obtained cocrystals of VDR-LBD with analogs 1-4. X-ray crystallog. anal. showed a covalent bond with His301 in VDR-LBD. We successfully synthesized vitamin D analogs that form a covalent bond with VDR-LBD.
- 340Liu, S.; Widom, J.; Kemp, C. W.; Crews, C. M.; Clardy, J. Structure of Human Methionine Aminopeptidase-2 Complexed with Fumagillin. Science 1998, 282 (5392), 1324– 1327, DOI: 10.1126/science.282.5392.1324[Crossref], [PubMed], [CAS], Google Scholar340https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXnsFenurY%253D&md5=729e37df06b4055f246753a544b6ee18Structure of human methionine aminopeptidase-2 complexed with fumagillinLiu, Shenping; Widom, Joanne; Kemp, Christopher W.; Crews, Craig M.; Clardy, JonScience (Washington, D. C.) (1998), 282 (5392), 1324-1327CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The fungal metabolite fumagillin suppresses the formation of new blood vessels, and a fumagillin analog is currently in clin. trials as an anticancer agent. The mol. target of fumagillin is methionine aminopeptidase-2 (MetAP-2). A 1.8 Å resoln. crystal structure of free and inhibited human MetAP-2 shows a covalent bond formed between a reactive epoxide of fumagillin and histidine-231 in the active site of MetAP-2. Extensive hydrophobic and water-mediated polar interactions with other parts of fumagillin provide addnl. affinity. Fumagillin-based drugs inhibit MetAP-2 but not MetAP-1, and the three-dimensional structure also indicates the likely determinants of this specificity. The structural basis for fumagillin's potency and specificity forms the starting point for structure-based drug design.
- 341Morgen, M.; Jöst, C.; Malz, M.; Janowski, R.; Niessing, D.; Klein, C. D.; Gunkel, N.; Miller, A. K. Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular Activity. ACS Chem. Biol. 2016, 11 (4), 1001– 1011, DOI: 10.1021/acschembio.5b00755[ACS Full Text], [CAS], Google Scholar341https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVGitbvO&md5=f83da1a8a3c4717b16cd0e90cc05f8a8Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular ActivityMorgen, Michael; Joest, Christian; Malz, Mona; Janowski, Robert; Niessing, Dierk; Klein, Christian D.; Gunkel, Nikolas; Miller, Aubry K.ACS Chemical Biology (2016), 11 (4), 1001-1011CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Methionine aminopeptidases (MetAPs) are responsible for the cotranslational cleavage of initiator methionines from nascent proteins. The MetAP2 subtype is up-regulated in many cancers, and selective inhibition of MetAP2 suppresses both vascularization and growth of tumors in animal models. The natural product fumagillin is a selective and potent irreversible inhibitor of MetAP2, and semisynthetic derivs. of fumagillin have shown promise in clin. studies for the treatment of cancer, and, more recently, for obesity. Further development of fumagillin derivs. has been complicated, however, by their generally poor pharmacokinetics. In an attempt to overcome these limitations, we developed an easily diversifiable synthesis of a novel class of MetAP2 inhibitors that were designed to mimic fumagillin's mol. scaffold but have improved pharmacol. profiles. These substances were found to be potent and selective inhibitors of MetAP2, as demonstrated in biochem. enzymic assays against three MetAP isoforms. Inhibitors with the same relative and abs. stereoconfiguration as fumagillin displayed significantly higher activity than their diastereomeric and enantiomeric isomers. X-ray crystallog. anal. revealed that the inhibitors covalently modify His231 in the MetAP2 active site via ring-opening of a spiroepoxide. Biochem. active substances inhibited the growth of endothelial cells and a MetAP2-sensitive cancer cell line, while closely related inactive isomers had little effect on the proliferation of either cell type. These effects correlated with altered N-terminal processing of the protein 14-3-3-γ. Finally, selected substances were found to have improved stabilities in mouse plasma and microsomes relative to the clin. investigated fumagillin deriv. beloranib.
- 342Jakob, C. G.; Upadhyay, A. K.; Donner, P. L.; Nicholl, E.; Addo, S. N.; Qiu, W.; Ling, C.; Gopalakrishnan, S. M.; Torrent, M.; Cepa, S. P.; Shanley, J.; Shoemaker, A. R.; Sun, C. C.; Vasudevan, A.; Woller, K. R.; Shotwell, J. B.; Shaw, B.; Bian, Z.; Hutti, J. E. Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315. J. Med. Chem. 2018, 61 (15), 6647– 6657, DOI: 10.1021/acs.jmedchem.8b00305[ACS Full Text], [CAS], Google Scholar342https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlWrtrbF&md5=15c54988cd3080d5a675badbb62b6fecNovel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315Jakob, Clarissa G.; Upadhyay, Anup K.; Donner, Pamela L.; Nicholl, Emily; Addo, Sadiya N.; Qiu, Wei; Ling, Christopher; Gopalakrishnan, Sujatha M.; Torrent, Maricel; Cepa, Steven P.; Shanley, Jason; Shoemaker, Alexander R.; Sun, Chaohong C.; Vasudevan, Anil; Woller, Kevin R.; Shotwell, J. Brad; Shaw, Bailin; Bian, Zhiguo; Hutti, Jessica E.Journal of Medicinal Chemistry (2018), 61 (15), 6647-6657CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)IDH1 plays a crit. role in a no. of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochem. characterization of two novel inhibitors of wild-type IDH1. In addn., we present the first ligand-bound crystallog. characterization of these novel small mol. IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsatd. enone 1 makes a unique covalent linkage through active site H315. As few small mols. have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small mol. design.
- 343Lin, S.; Yang, X.; Jia, S.; Weeks, A. M.; Hornsby, M.; Lee, P. S.; Nichiporuk, R. V.; Iavarone, A. T.; Wells, J. A.; Toste, F. D.; Chang, C. J. Redox-Based Reagents for Chemoselective Methionine Bioconjugation. Science 2017, 355 (6325), 597– 602, DOI: 10.1126/science.aal3316[Crossref], [PubMed], [CAS], Google Scholar343https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXit1Okur4%253D&md5=90957bb9f95794e12417ade39bebd734Redox-based reagents for chemoselective methionine bioconjugationLin, Shixian; Yang, Xiaoyu; Jia, Shang; Weeks, Amy M.; Hornsby, Michael; Lee, Peter S.; Nichiporuk, Rita V.; Iavarone, Anthony T.; Wells, James A.; Toste, F. Dean; Chang, Christopher J.Science (Washington, DC, United States) (2017), 355 (6325), 597-602CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In contrast, selective ligation to the other sulfur-contg. amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, the authors report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. The authors highlight the broad utility of this conjugation method to enable precise addn. of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes.
- 344Bizet, V.; Hendriks, C. M. M.; Bolm, C. Sulfur Imidations: Access to Sulfimides and Sulfoximines. Chem. Soc. Rev. 2015, 44 (11), 3378– 3390, DOI: 10.1039/C5CS00208G[Crossref], [PubMed], [CAS], Google Scholar344https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXns1yisrw%253D&md5=f783729be2df3166e6e3b09c3b6d311fSulfur imidations: access to sulfimides and sulfoximinesBizet, Vincent; Hendriks, Christine M. M.; Bolm, CarstenChemical Society Reviews (2015), 44 (11), 3378-3390CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Being mono-aza analogs of sulfoxides and sulfones, sulfimides and sulfoximines, resp., are important compds. in asym. synthesis, crop protection and medicinal chem. various methods were developed for their prepn. For their prepn. various methods were developed. In the search for the optimal synthetic approach for a given target compd., several parameters have to be considered which also include safety issues and availability of starting materials. An overview of sulfur imidation methods, classified by imidating agents and compds. with a related behavior were reviewed. The aim of this survey was to provide a practical ''tool box'' for the synthetic chemist by mapping the advantages and disadvantages assocd. with the use of these compds.
- 345Gong, Y.; Andina, D.; Nahar, S.; Leroux, J.-C.; Gauthier, M. A. Releasable and Traceless PEGylation of Arginine-Rich Antimicrobial Peptides. Chem. Sci. 2017, 8 (5), 4082– 4086, DOI: 10.1039/C7SC00770A[Crossref], [PubMed], [CAS], Google Scholar345https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsVakur4%253D&md5=ce26e313c6d8779e5c48a86d80eb47efReleasable and traceless PEGylation of arginine-rich antimicrobial peptidesGong, Y.; Andina, D.; Nahar, S.; Leroux, J.-C.; Gauthier, M. A.Chemical Science (2017), 8 (5), 4082-4086CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Arginine-rich antimicrobial peptides (AMPs) are emerging therapeutics of interest. However, their applicability is limited by their short circulation half-life, caused in part by their small size and digestion by blood proteases. This study reports a strategy to temporarily mask arginine residues within AMPs with methoxy poly(ethylene glycol). Based on the reagent used, release of AMPs occurred in hours to days in a completely traceless fashion. In vitro, conjugates were insensitive to serum proteases, and released native AMP with full in vitro bioactivity. This strategy is thus highly relevant and should be adaptable to the entire family of arginine-rich AMPs. It may potentially be used to improve AMP-therapies by providing a more steady concn. of AMP in the blood after a single injection, avoiding toxic effects at high AMP doses, and reducing the no. of doses required over the treatment duration.
- 346Seki, Y.; Ishiyama, T.; Sasaki, D.; Abe, J.; Sohma, Y.; Oisaki, K.; Kanai, M. Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins. J. Am. Chem. Soc. 2016, 138 (34), 10798– 10801, DOI: 10.1021/jacs.6b06692[ACS Full Text], [CAS], Google Scholar346https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlOgt7%252FO&md5=1dd578dc20df2d1092694c474a4c2c4dTransition Metal-Free Tryptophan-Selective Bioconjugation of ProteinsSeki, Yohei; Ishiyama, Takashi; Sasaki, Daisuke; Abe, Junpei; Sohma, Youhei; Oisaki, Kounosuke; Kanai, MotomuJournal of the American Chemical Society (2016), 138 (34), 10798-10801CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Chem. modifications of native proteins can facilitate prodn. of supernatural protein functions that are not easily accessible by complementary methods relying on genetic manipulations. However, accomplishing precise control over selectivity while maintaining structural integrity and homogeneity still represents a formidable challenge. Herein, the authors report a transition metal-free method for tryptophan-selective bioconjugation of proteins that is based on an organoradical and operates under ambient conditions. This method exhibits low levels of cross-reactivity and leaves higher-order structures of the protein and various functional groups therein unaffected. The strategy to target less abundant amino acids contributes to the formation of structurally homogeneous conjugates, which may even be suitable for protein crystallog. The absence of toxic metals and biochem. incompatible conditions allows a rapid functional modulation of native proteins such as antibodies and pathogenic aggregative proteins, and this method may thus easily find therapeutic applications.
- 347Shibata, Y.; Chiba, M. The Role of Extrahepatic Metabolism in the Pharmacokinetics of the Targeted Covalent Inhibitors Afatinib, Ibrutinib, and Neratinib. Drug Metab. Dispos. 2015, 43 (3), 375– 384, DOI: 10.1124/dmd.114.061424[Crossref], [PubMed], [CAS], Google Scholar347https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXit1OmsrY%253D&md5=eaac67ce7cea5f2432c5144922ba3dd2The role of extrahepatic metabolism in the pharmacokinetics of the targeted covalent inhibitors afatinib, ibrutinib, and neratinibShibata, Yoshihiro; Chiba, MasatoDrug Metabolism & Disposition (2015), 43 (3), 375-384, 10 pp.CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)Despite the fact that much progress has been made recently in the development of targeted covalent inhibitors (TCIs), their pharmacokinetics (PK) have not been well characterized in the light of extrahepatic clearance (CLextH) by glutathione (GSH)/glutathione S-transferase (GST)-dependent conjugation attributable to the unique electrophilic structure (e.g., acrylamide moiety) of TCI compds. In the present study, CLextH values were examd. in rat, dog, and monkey to predict the contribution of CLextH to the PK of the TCIs afatinib, ibrutinib, and neratinib in humans. Afatinib and neratinib both underwent extensive conjugation with GSH in buffer and cytosol fractions of liver and kidney, whereas ibrutinib showed much lower reactivity/susceptibility to GSH/GST-dependent conjugation. The CLextH in each species was calcd. from the difference between obsd. total body clearance and predicted hepatic clearance (CLH) in cryopreserved hepatocytes suspended in 100% serum of the corresponding species. The power-based simple allometry relating the CLextH for the unbound compd. to animal body wt. was applicable across species for afatinib and neratinib (R2 ≥ 0.9) but not for ibrutinib (R2 = 0.04). The predicted AUC after oral administration of afatinib and neratinib agreed reasonably closely with reported values in phase I dose-escalation studies. Comparisons of CLextH and CLH predicted that CLextH largely detd. the PK of afatinib (>90% as a proportion of total body clearance) and neratinib (∼34%) in humans. The present method can serve as one of the tools for the optimization of PK in humans at the discovery stage for the development of TCI candidates.
- 348Scheers, E.; Leclercq, L.; de Jong, J.; Bode, N.; Bockx, M.; Laenen, A.; Cuyckens, F.; Skee, D.; Murphy, J.; Sukbuntherng, J.; Mannens, G. Absorption, Metabolism, and Excretion of Oral 14C Radiolabeled Ibrutinib: An Open-Label, Phase I, Single-Dose Study in Healthy Men. Drug Metab. Dispos. 2015, 43, 289– 297, DOI: 10.1124/dmd.114.060061[Crossref], [PubMed], [CAS], Google Scholar348https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpt1ansg%253D%253D&md5=1f1e728078220c9d7cd973d4992c7308Absorption, metabolism, and excretion of pral 14C radiolabeled ibrutinib: an open-label, phase I, single-dose study in healthy menScheers, Ellen; Leclercq, Laurent; de Jong, Jan; Bode, Nini; Bockx, Marc; Laenen, Aline; Cuyckens, Filip; Skee, Donna; Murphy, Joe; Sukbuntherng, Juthamas; Mannens, GeertDrug Metabolism & Disposition (2015), 43 (2), 289-297, 9 pp.CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)The absorption, metab., and excretion of ibrutinib were investigated in healthy men after administration of a single oral dose of 140 mg of 14C-labeled ibrutinib. The mean S.D. cumulative excretion of radioactivity of the dose was 7.8% 1.4% in urine and 80.6% 3.1% in feces with <1% excreted as parent ibrutinib. Only oxidative metabolites and very limited parent compd. were detected in feces, and this indicated that ibrutinib was completely absorbed from the gastrointestinal tract. Metab. occurred via three major pathways hydroxylation of the Ph M35, opening of the piperidine M25 and M34, and epoxidn. of the ethylene on the acryloyl moiety with further hydrolysis to dihydrodiol PCI-45227, and M37. Addnl. metabolites were formed by combinations of the primary metabolic pathways or by further metab. In blood and plasma, a rapid initial decline in radioactivity was obsd. along with long terminal elimination half-life for total radioactivity. The max. concn. Cmax and area under the concn.-time curve AUC for total radioactivity were higher in plasma compared with blood. The main circulating entities in blood and plasma were M21 sulfate conjugate of a monooxidized metabolite on phenoxyphenyl, M25, M34, M37 PCI-45227, and ibrutinib. At Cmax of radioactivity, 12% of total radioactivity was accounted for by covalent binding in human plasma. More than 50% of total plasma radioactivity was attributed to covalently bound material from 8 h onward; as a result, covalent binding accounted for 38% and 51% of total radioactivity AUC0-24 h and AUC0-72 h, resp. No effect of CYP2D6 genotype was obsd. on ibrutinib metab. Ibrutinib was well-tolerated by healthy participants.
- 349Chatterjee, P.; Botello-Smith, W. M.; Zhang, H.; Qian, L.; Alsamarah, A.; Kent, D.; Lacroix, J. J.; Baudry, M.; Luo, Y. Can Relative Binding Free Energy Predict Selectivity of Reversible Covalent Inhibitors?. J. Am. Chem. Soc. 2017, 139 (49), 17945– 17952, DOI: 10.1021/jacs.7b08938[ACS Full Text], [CAS], Google Scholar349https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslyrs7bI&md5=e6a38ff570134e956eb43bd4a874ca55Can Relative Binding Free Energy Predict Selectivity of Reversible Covalent Inhibitors?Chatterjee, Payal; Botello-Smith, Wesley M.; Zhang, Han; Qian, Li; Alsamarah, Abdelaziz; Kent, David; Lacroix, Jerome J.; Baudry, Michel; Luo, YunJournal of the American Chemical Society (2017), 139 (49), 17945-17952CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Reversible covalent inhibitors have many clin. advantages over noncovalent or irreversible covalent drugs. However, apart from selecting a warhead, substantial efforts in design and synthesis are needed to optimize noncovalent interactions to improve target-selective binding. Computational prediction of binding affinity for reversible covalent inhibitors presents a unique challenge since the binding process consists of multiple steps, which are not necessarily independent of each other. In this study, the authors lay out the relation between relative binding free energy and the overall reversible covalent binding affinity using a two-state binding model. To prove the concept, the authors employed free energy perturbation (FEP) coupled with λ-exchange mol. dynamics method to calc. the binding free energy of a series of α-ketoamide analogs relative to a common warhead scaffold, in both noncovalent and covalent binding states, and for two highly homologous proteases, calpain-1 and calpain-2. The authors conclude that covalent binding state alone, in general, can be used to predict reversible covalent binding selectivity. However, exceptions may exist. Therefore, the authors also discuss the conditions under which the noncovalent binding step is no longer negligible and propose to combine the relative FEP calcns. with a single QM/MM calcn. of warhead to predict the binding affinity and binding kinetics. The FEP calcns. also revealed that covalent and noncovalent binding states of an inhibitor do not necessarily exhibit the same selectivity. Thus, investigating both binding states, as well as the kinetics will provide extremely useful information for optimizing reversible covalent inhibitors.
- 350Alberty, R. A.; Hammes, G. G. Application of the Theory of Diffusion-Controlled Reactions to Enzyme Kinetics. J. Phys. Chem. 1958, 62 (2), 154– 159, DOI: 10.1021/j150560a005[ACS Full Text], [CAS], Google Scholar350https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG1cXlsVSqtg%253D%253D&md5=140ac5d97b80b78b09c1a8523a7c1df7Application of the theory of diffusion-controlled reactions to enzyme kineticsAlberty, Robert A.; Hammes, Gordon G.Journal of Physical Chemistry (1958), 62 (), 154-9CODEN: JPCHAX; ISSN:0022-3654.cf. C.A. 51, 9757h. Extrapolation of the min. values of the 2nd-order rate consts. for the reaction of fumarate ions with fumarase to zero ionic strength yields a value of about 3 × 1010/sec. mole at 25°. The magnitude of this rate const. can be accounted for theoretically by considering the diffusion of substrate into a hemispherical reaction site on a plane with a reaction radius of 5 A., provided that the effect of elec. charges in increasing the rate is included. It is presumed that the bimol. reaction of fumarase with its substrates is diffusion controlled. The effect of electrolyte concn. on the experimentally detd. min. values of the 2nd-order rate consts. indicates a charge of +2 to +3 on the enzymic site. The application of these theoretical considerations to enzymic reactions, in general, is discussed for the case where the effect of protein charges outside the enzymic site may be neglected.
- 351Wright, M. H.; Sieber, S. A. Chemical Proteomics Approaches for Identifying the Cellular Targets of Natural Products. Nat. Prod. Rep. 2016, 33 (5), 681– 708, DOI: 10.1039/C6NP00001K[Crossref], [PubMed], [CAS], Google Scholar351https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsF2gsbc%253D&md5=0732834925092e013d9545f46ce4fd2fChemical proteomics approaches for identifying the cellular targets of natural productsWright, M. H.; Sieber, S. A.Natural Product Reports (2016), 33 (5), 681-708CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)Covering: 2010 up to 2016: Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chem. and biol. today. Chem. proteomics is a growing area of chem. biol. that seeks to design small mol. probes to understand protein function. In the context of natural products, chem. proteomics can be used to identify the protein binding partners or targets of small mols. in live cells. Here, the authors highlight recent examples of chem. probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chem. reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. The authors also focus here on strategies that employ a click reaction, the copper-catalyzed azide-alkyne cycloaddn. reaction (CuAAC), to allow minimal functionalization of natural product scaffolds with an alkyne or azide tag. The authors also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterized chem. probe for binding to a particular set of protein targets. Fueled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quant. proteomics to help define the true interacting partners of probes, and the authors highlight the opportunities this rapidly evolving technol. provides in chem. proteomics. Finally, some of the limitations and challenges of chem. proteomics approaches are discussed.
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(5)
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(1)
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(23)
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(21)
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(21)
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(20)
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(15)
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(13)
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(13)
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(6)
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(11)
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(23)
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- Jing Leng, Wenjian Tang, Wan-Yin Fang, Chuang Zhao, Hua-Li Qin. A Simple Protocol for the Stereoselective Construction of Enaminyl Sulfonyl Fluorides. Organic Letters 2020, 22
(11)
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(8)
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(7)
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(52)
, 5234-5244. https://doi.org/10.1021/acs.biochem.9b00293
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(51)
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(23)
, 10497-10525. https://doi.org/10.1021/acs.jmedchem.9b00683
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(11)
, 10477-10488. https://doi.org/10.1021/acscatal.9b03640
- Angus Voice, Gary Tresadern, Herman van Vlijmen, Adrian Mulholland. Limitations of Ligand-Only Approaches for Predicting the Reactivity of Covalent Inhibitors. Journal of Chemical Information and Modeling 2019, 59
(10)
, 4220-4227. https://doi.org/10.1021/acs.jcim.9b00404
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(20)
, 9188-9200. https://doi.org/10.1021/acs.jmedchem.9b01108
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(17)
, 7874-7884. https://doi.org/10.1021/acs.jmedchem.9b00642
- Ferruccio Palazzesi, Marc A. Grundl, Alexander Pautsch, Alexander Weber, Christofer S. Tautermann. A Fast Ab Initio Predictor Tool for Covalent Reactivity Estimation of Acrylamides. Journal of Chemical Information and Modeling 2019, 59
(8)
, 3565-3571. https://doi.org/10.1021/acs.jcim.9b00316
- Laurent Hoffer, Magali Saez-Ayala, Dragos Horvath, Alexandre Varnek, Xavier Morelli, Philippe Roche. CovaDOTS: In Silico Chemistry-Driven Tool to Design Covalent Inhibitors Using a Linking Strategy. Journal of Chemical Information and Modeling 2019, 59
(4)
, 1472-1485. https://doi.org/10.1021/acs.jcim.8b00960
- Kangsa Amporndanai, Xiaoli Meng, Weijuan Shang, Zhenmig Jin, Michael Rogers, Yao Zhao, Zihe Rao, Zhi-Jie Liu, Haitao Yang, Leike Zhang, Paul M. O’Neill, S. Samar Hasnain. Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives. Nature Communications 2021, 12
(1)
https://doi.org/10.1038/s41467-021-23313-7
- Haixia Su, Sheng Yao, Wenfeng Zhao, Yumin Zhang, Jia Liu, Qiang Shao, Qingxing Wang, Minjun Li, Hang Xie, Weijuan Shang, Changqiang Ke, Lu Feng, Xiangrui Jiang, Jingshan Shen, Gengfu Xiao, Hualiang Jiang, Leike Zhang, Yang Ye, Yechun Xu. Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease. Nature Communications 2021, 12
(1)
https://doi.org/10.1038/s41467-021-23751-3
- Xian Zhou, Xuexin Feng, Dachi Wang, Deheng Chen, Gaoxing Wu, Ziqin Yan, Xilin Lyu, Huan Wang, Jin-Ming Yang, Yujun Zhao. Synthesis and bioactivity studies of covalent inhibitors derived from (-)-Chaetominine. Journal of Molecular Structure 2021, 1241 , 130694. https://doi.org/10.1016/j.molstruc.2021.130694
- S.A. Adediran, Michael J. Morrison, R.F. Pratt. Detection of an enzyme isomechanism by means of the kinetics of covalent inhibition. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2021, 1869
(9)
, 140681. https://doi.org/10.1016/j.bbapap.2021.140681
- Zhi-Zheng Wang, Xing-Xing Shi, Guang-Yi Huang, Ge-Fei Hao, Guang-Fu Yang. Fragment-based drug design facilitates selective kinase inhibitor discovery. Trends in Pharmacological Sciences 2021, 42
(7)
, 551-565. https://doi.org/10.1016/j.tips.2021.04.001
- Huabin Hu, Jürgen Bajorath. Systematic assessment of structure-promiscuity relationships between different types of kinase inhibitors. Bioorganic & Medicinal Chemistry 2021, 41 , 116226. https://doi.org/10.1016/j.bmc.2021.116226
- Muya Xiong, Haixia Su, Wenfeng Zhao, Hang Xie, Qiang Shao, Yechun Xu. What coronavirus 3C‐like protease tells us: From structure, substrate selectivity, to inhibitor design. Medicinal Research Reviews 2021, 41
(4)
, 1965-1998. https://doi.org/10.1002/med.21783
- John P. Guilinger, Archna Archna, Martin Augustin, Andreas Bergmann, Paolo A. Centrella, Matthew A. Clark, John W. Cuozzo, Maike Däther, Marie-Aude Guié, Sevan Habeshian, Reiner Kiefersauer, Stephan Krapp, Alfred Lammens, Lukas Lercher, Julie Liu, Yanbin Liu, Klaus Maskos, Michael Mrosek, Klaus Pflügler, Markus Siegert, Heather A. Thomson, Xia Tian, Ying Zhang, Debora L. Konz Makino, Anthony D. Keefe. Novel irreversible covalent BTK inhibitors discovered using DNA-encoded chemistry. Bioorganic & Medicinal Chemistry 2021, 42 , 116223. https://doi.org/10.1016/j.bmc.2021.116223
- Yaping Cheng, Jingyuan Wu, Ying Han, Jingyao Xu, Yifan Da, Qian Zhao, Guoying Guo, Yani Zhou, Yimin Chen, Jinghong Liu, Huayao Chen, Xianxing Jiang, Xiaoqing Cai. A CDR-based approach to generate covalent inhibitory antibody for human rhinovirus protease. Bioorganic & Medicinal Chemistry 2021, 42 , 116219. https://doi.org/10.1016/j.bmc.2021.116219
- David Quach, Guanghui Tang, Jothi Anantharajan, Nithya Baburajendran, Anders Poulsen, John L. K. Wee, Priya Retna, Rong Li, Boping Liu, Doris H. Y. Tee, Perlyn Z. Kwek, Joma K. Joy, Wan‐Qi Yang, Chong‐Jing Zhang, Klement Foo, Thomas H. Keller, Shao Q. Yao. Strategic Design of Catalytic Lysine‐Targeting Reversible Covalent BCR‐ABL Inhibitors**. Angewandte Chemie 2021, 319 https://doi.org/10.1002/ange.202105383
- Jeffrey G. Martin, Jennifer A. Ward, Felix Feyertag, Lu Zhang, Shalise Couvertier, Kevin Guckian, Kilian V. M. Huber, Douglas S. Johnson. Chemoproteomic Profiling of Covalent XPO1 Inhibitors to Assess Target Engagement and Selectivity. ChemBioChem 2021, 22
(12)
, 2116-2123. https://doi.org/10.1002/cbic.202100038
- Tao Wang, Yanqing Wang, Kai Xu, Yuheng Zhang, Jiarui Guo, Lantao Liu. Transition‐Metal‐Free DMAP‐Mediated Aromatic Esterification of Amides with Organoboronic Acids. European Journal of Organic Chemistry 2021, 2021
(22)
, 3274-3277. https://doi.org/10.1002/ejoc.202100478
- Yaniv Tivon, Gianna Falcone, Alexander Deiters. Protein Labeling and Crosslinking by Covalent Aptamers. Angewandte Chemie International Edition 2021, 4 https://doi.org/10.1002/anie.202101174
- Yaniv Tivon, Gianna Falcone, Alexander Deiters. Protein Labeling and Crosslinking by Covalent Aptamers. Angewandte Chemie 2021, 4 https://doi.org/10.1002/ange.202101174
- Yudai Tabuchi, Takahito Watanabe, Riku Katsuki, Yuji Ito, Masumi Taki. Direct screening of a target-specific covalent binder: stringent regulation of warhead reactivity in a matchmaking environment. Chemical Communications 2021, 57
(44)
, 5378-5381. https://doi.org/10.1039/D1CC01773J
- Matthew D. Lloyd, Maksims Yevglevskis, Amit Nathubhai, Tony D. James, Michael D. Threadgill, Timothy J. Woodman. Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition. Chemical Society Reviews 2021, 50
(10)
, 5952-5984. https://doi.org/10.1039/D0CS00540A
- Matthias Franz, Britta Mörchen, Carsten Degenhart, Daniel Gülden, Oleksandr Shkura, Dirk Wolters, Uwe Koch, Bert Klebl, Raphael Stoll, Iris Helfrich, Jürgen Scherkenbeck. Sequence‐Selective Covalent CaaX‐Box Receptors Prevent Farnesylation of Oncogenic Ras Proteins and Impact MAPK/PI3 K Signaling. ChemMedChem 2021, 94 https://doi.org/10.1002/cmdc.202100167
- David Quach, Guanghui Tang, Jothi Anantharajan, Nithya Baburajendran, Anders Poulsen, John Wee, Priya Retna, Rong Li, Boping Liu, Doris Tee, Perlyn Kwek, Joma Joy, Wang-Qi Yang, Chong-Jing Zhang, Klement Foo, Thomas Keller, Shao Q. Yao. Strategic Design of Catalytic Lysine‐Targeting Reversible Covalent BCR‐ABL Inhibitors. Angewandte Chemie International Edition 2021, https://doi.org/10.1002/anie.202105383
- Kewei Sun, Zhonghao Sun, Fenglan Zhao, Guangzhi Shan, Qingguo Meng. Recent advances in research of colchicine binding site inhibitors and their interaction modes with tubulin. Future Medicinal Chemistry 2021, 13
(9)
, 839-858. https://doi.org/10.4155/fmc-2020-0376
- Miljan Kuljanin, Dylan C. Mitchell, Devin K. Schweppe, Ajami S. Gikandi, David P. Nusinow, Nathan J. Bulloch, Ekaterina V. Vinogradova, David L. Wilson, Eric T. Kool, Joseph D. Mancias, Benjamin F. Cravatt, Steven P. Gygi. Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries. Nature Biotechnology 2021, 39
(5)
, 630-641. https://doi.org/10.1038/s41587-020-00778-3
- Ivy Guan, Kayla Williams, Jolyn Pan, Xuyu Liu. New Cysteine Covalent Modification Strategies Enable Advancement of Proteome‐wide Selectivity of Kinase Modulators. Asian Journal of Organic Chemistry 2021, 10
(5)
, 949-963. https://doi.org/10.1002/ajoc.202100036
- Julian Breidenbach, Carina Lemke, Thanigaimalai Pillaiyar, Laura Schäkel, Ghazl Al Hamwi, Miriam Diett, Robin Gedschold, Nina Geiger, Vittoria Lopez, Salahuddin Mirza, Vigneshwaran Namasivayam, Anke C. Schiedel, Katharina Sylvester, Dominik Thimm, Christin Vielmuth, Lan Phuong Vu, Maria Zyulina, Jochen Bodem, Michael Gütschow, Christa E. Müller. Die Hauptprotease von SARS‐CoV‐2 als Zielstruktur: Von der Etablierung eines Hochdurchsatz‐Screenings zum Design maßgeschneiderter Inhibitoren. Angewandte Chemie 2021, 133
(18)
, 10515-10521. https://doi.org/10.1002/ange.202016961
- Julian Breidenbach, Carina Lemke, Thanigaimalai Pillaiyar, Laura Schäkel, Ghazl Al Hamwi, Miriam Diett, Robin Gedschold, Nina Geiger, Vittoria Lopez, Salahuddin Mirza, Vigneshwaran Namasivayam, Anke C. Schiedel, Katharina Sylvester, Dominik Thimm, Christin Vielmuth, Lan Phuong Vu, Maria Zyulina, Jochen Bodem, Michael Gütschow, Christa E. Müller. Targeting the Main Protease of SARS‐CoV‐2: From the Establishment of High Throughput Screening to the Design of Tailored Inhibitors. Angewandte Chemie International Edition 2021, 60
(18)
, 10423-10429. https://doi.org/10.1002/anie.202016961
- Kirandeep Samby, Paul A. Willis, Jeremy N. Burrows, Benoît Laleu, Peter J. H. Webborn, . Actives from MMV Open Access Boxes? A suggested way forward. PLOS Pathogens 2021, 17
(4)
, e1009384. https://doi.org/10.1371/journal.ppat.1009384
- Angus T. Voice, Gary Tresadern, Rebecca M. Twidale, Herman van Vlijmen, Adrian J. Mulholland. Mechanism of covalent binding of ibrutinib to Bruton's tyrosine kinase revealed by QM/MM calculations. Chemical Science 2021, 12
(15)
, 5511-5516. https://doi.org/10.1039/D0SC06122K
- Clinton G. L. Veale. Into the Fray! A Beginner's Guide to Medicinal Chemistry. ChemMedChem 2021, 16
(8)
, 1199-1225. https://doi.org/10.1002/cmdc.202000929
- Jiamin Zheng, Jun Wu, Xiao Ding, Hong C. Shen, Ge Zou. Small molecule approaches to treat autoimmune and inflammatory diseases (Part I): Kinase inhibitors. Bioorganic & Medicinal Chemistry Letters 2021, 38 , 127862. https://doi.org/10.1016/j.bmcl.2021.127862
- Tasuku Ishida, Alessio Ciulli. E3 Ligase Ligands for PROTACs: How They Were Found and How to Discover New Ones. SLAS DISCOVERY: Advancing the Science of Drug Discovery 2021, 26
(4)
, 484-502. https://doi.org/10.1177/2472555220965528
- Hong-Ru Chen, Zhen-Yu Hu, Hua-Li Qin, Haolin Tang. A novel three-component reaction for constructing indolizine-containing aliphatic sulfonyl fluorides. Organic Chemistry Frontiers 2021, 8
(6)
, 1185-1189. https://doi.org/10.1039/D0QO01430C
- Thomas A. Baillie. Approaches to mitigate the risk of serious adverse reactions in covalent drug design. Expert Opinion on Drug Discovery 2021, 16
(3)
, 275-287. https://doi.org/10.1080/17460441.2021.1832079
- Robert Roskoski. Orally effective FDA-approved protein kinase targeted covalent inhibitors (TCIs). Pharmacological Research 2021, 165 , 105422. https://doi.org/10.1016/j.phrs.2021.105422
- Valentin Wydra, Stefan Gerstenecker, Dieter Schollmeyer, Stanislav Andreev, Teodor Dimitrov, Ricardo Augusto Massarico Serafim, Stefan Laufer, Matthias Gehringer. N-(6-Chloro-3-nitropyridin-2-yl)-5-(1-methyl-1H-pyrazol-4-yl)isoquinolin-3-amine. Molbank 2021, 2021
(1)
, M1181. https://doi.org/10.3390/M1181
- Paweł Łukasik, Irena Baranowska-Bosiacka, Katarzyna Kulczycka, Izabela Gutowska. Inhibitors of Cyclin-Dependent Kinases: Types and Their Mechanism of Action. International Journal of Molecular Sciences 2021, 22
(6)
, 2806. https://doi.org/10.3390/ijms22062806
- Samuel Ofori, Sailajah Gukathasan, Samuel G. Awuah. Gold‐Based Pharmacophore Inhibits Intracellular MYC Protein. Chemistry – A European Journal 2021, 27
(12)
, 4168-4175. https://doi.org/10.1002/chem.202004962
- Borvornwat Toviwek, Duangkamol Gleeson, M. Paul Gleeson. QM/MM and molecular dynamics investigation of the mechanism of covalent inhibition of TAK1 kinase. Organic & Biomolecular Chemistry 2021, 19
(6)
, 1412-1425. https://doi.org/10.1039/D0OB02273J
- Rita Fuerst, Rolf Breinbauer. Activity‐Based Protein Profiling (ABPP) of Oxidoreductases. ChemBioChem 2021, 22
(4)
, 630-638. https://doi.org/10.1002/cbic.202000542
- László Petri, Péter Ábrányi‐Balogh, Imre Tímea, Gyula Pálfy, András Perczel, Damijan Knez, Martina Hrast, Martina Gobec, Izidor Sosič, Kinga Nyíri, Beáta G. Vértessy, Niklas Jänsch, Charlotte Desczyk, Franz‐Josef Meyer‐Almes, Iza Ogris, Simona Golič Grdadolnik, Luca Giacinto Iacovino, Claudia Binda, Stanislav Gobec, György M. Keserű. Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments. ChemBioChem 2021, 22
(4)
, 743-753. https://doi.org/10.1002/cbic.202000700
- Kemel Arafet, Natalia Serrano-Aparicio, Alessio Lodola, Adrian J. Mulholland, Florenci V. González, Katarzyna Świderek, Vicent Moliner. Mechanism of inhibition of SARS-CoV-2 M
pro
by N3 peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity. Chemical Science 2021, 12
(4)
, 1433-1444. https://doi.org/10.1039/D0SC06195F
- Lei Wang, Louis P. Riel, Bekim Bajrami, Bin Deng, Amy R. Howell, Xudong Yao. α‐Methylene‐β‐Lactone Scaffold for Developing Chemical Probes at the Two Ends of the Selectivity Spectrum. ChemBioChem 2021, 22
(3)
, 505-515. https://doi.org/10.1002/cbic.202000605
- Sun Dongbang, Jonathan A. Ellman. Synthesis of Nitrile Bearing Acyclic Quaternary Centers through Co(III)‐Catalyzed Sequential C−H Bond Addition to Dienes and
N
‐Cyanosuccinimide. Angewandte Chemie 2021, 133
(4)
, 2163-2167. https://doi.org/10.1002/ange.202010735
- Sun Dongbang, Jonathan A. Ellman. Synthesis of Nitrile Bearing Acyclic Quaternary Centers through Co(III)‐Catalyzed Sequential C−H Bond Addition to Dienes and
N
‐Cyanosuccinimide. Angewandte Chemie International Edition 2021, 60
(4)
, 2135-2139. https://doi.org/10.1002/anie.202010735
- Hongyan Du, Junbo Gao, Gaoqi Weng, Junjie Ding, Xin Chai, Jinping Pang, Yu Kang, Dan Li, Dongsheng Cao, Tingjun Hou. CovalentInDB: a comprehensive database facilitating the discovery of covalent inhibitors. Nucleic Acids Research 2021, 49
(D1)
, D1122-D1129. https://doi.org/10.1093/nar/gkaa876
- Changyu Huang, Jinpeng Li, Jiaquan Wang, Qingshu Zheng, Zhenhua Li, Tao Tu. Hydrogen-bond-assisted transition-metal-free catalytic transformation of amides to esters. Science China Chemistry 2021, 64
(1)
, 66-71. https://doi.org/10.1007/s11426-020-9883-3
- Richard A. Ward. Modeling Covalent Protein-Ligand Interactions. 2021,,, 174-189. https://doi.org/10.1016/B978-0-12-801238-3.11519-3
- Viktoriya Y. Berdan, Paul C. Klauser, Lei Wang. Covalent peptides and proteins for therapeutics. Bioorganic & Medicinal Chemistry 2021, 29 , 115896. https://doi.org/10.1016/j.bmc.2020.115896
- Elena De Vita. 10 years into the resurgence of covalent drugs. Future Medicinal Chemistry 2021, 13
(2)
, 193-210. https://doi.org/10.4155/fmc-2020-0236
- Solomon Tadesse, Derek R Duckett, Andrii Monastyrskyi. The promise and current status of CDK12/13 inhibition for the treatment of cancer. Future Medicinal Chemistry 2021, 13
(2)
, 117-141. https://doi.org/10.4155/fmc-2020-0240
- Lyn H. Jones. Target Validation—Prosecuting the Target. 2021,,https://doi.org/10.1016/B978-0-12-820472-6.00014-1
- Leonard Sung. Covalent drugs in development for immune-mediated diseases. 2021,,https://doi.org/10.1016/bs.armc.2021.03.001
- Hyunsoo Kim, Yoon Soo Hwang, Mingi Kim, Seung Bum Park. Recent advances in the development of covalent inhibitors. RSC Medicinal Chemistry 2021, 10 https://doi.org/10.1039/D1MD00068C
- Qingqing Dai, Yuhang Yan, Xiangli Ning, Gen Li, Junlin Yu, Ji Deng, Lingling Yang, Guo-Bo Li. AncPhore: A versatile tool for anchor pharmacophore steered drug discovery with applications in discovery of new inhibitors targeting metallo-β-lactamases and indoleamine/tryptophan 2,3-dioxygenases. Acta Pharmaceutica Sinica B 2021, 31 https://doi.org/10.1016/j.apsb.2021.01.018
- László Petri, Attila Egyed, Dávid Bajusz, Tímea Imre, Anasztázia Hetényi, Tamás Martinek, Péter Ábrányi-Balogh, György M. Keserű. An electrophilic warhead library for mapping the reactivity and accessibility of tractable cysteines in protein kinases. European Journal of Medicinal Chemistry 2020, 207 , 112836. https://doi.org/10.1016/j.ejmech.2020.112836
- Tobias Krojer, James S Fraser, Frank von Delft. Discovery of allosteric binding sites by crystallographic fragment screening. Current Opinion in Structural Biology 2020, 65 , 209-216. https://doi.org/10.1016/j.sbi.2020.08.004
- Giulia Bianco, David S. Goodsell, Stefano Forli. Selective and Effective: Current Progress in Computational Structure-Based Drug Discovery of Targeted Covalent Inhibitors. Trends in Pharmacological Sciences 2020, 41
(12)
, 1038-1049. https://doi.org/10.1016/j.tips.2020.10.005
- Tatu Pantsar. KRAS(G12C)–AMG 510 interaction dynamics revealed by all-atom molecular dynamics simulations. Scientific Reports 2020, 10
(1)
https://doi.org/10.1038/s41598-020-68950-y
- Leigh A. Stoddart, Nicholas D. Kindon, Omolade Otun, Clare R. Harwood, Foteini Patera, Dmitry B. Veprintsev, Jeanette Woolard, Stephen J. Briddon, Hester A. Franks, Stephen J. Hill, Barrie Kellam. Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells. Communications Biology 2020, 3
(1)
https://doi.org/10.1038/s42003-020-01451-w
- Michael Forster, Xiaojun Julia Liang, Martin Schröder, Stefan Gerstenecker, Apirat Chaikuad, Stefan Knapp, Stefan Laufer, Matthias Gehringer. Discovery of a Novel Class of Covalent Dual Inhibitors Targeting the Protein Kinases BMX and BTK. International Journal of Molecular Sciences 2020, 21
(23)
, 9269. https://doi.org/10.3390/ijms21239269
- Francesca Picarazzi, Ilaria Vicenti, Francesco Saladini, Maurizio Zazzi, Mattia Mori. Targeting the RdRp of Emerging RNA Viruses: The Structure-Based Drug Design Challenge. Molecules 2020, 25
(23)
, 5695. https://doi.org/10.3390/molecules25235695
- Shuai Liu, Yangen Huang, Xiu-Hua Xu, Feng-Ling Qing. Fluorosulfonylation of arenediazonium tetrafluoroborates with Na2S2O5 and N-fluorobenzenesulfonimide. Journal of Fluorine Chemistry 2020, 240 , 109653. https://doi.org/10.1016/j.jfluchem.2020.109653
Abstract
Figure 1
Figure 1. Kinetic selectivity of fumaric acid esters. Selectivity is povided by rapid bond formation with the target. Slightly slower ester cleavage deactivates the warhead, preventing even slower labeling of undesired proteins.
Figure 2
Figure 2. Ibrutinib-derived fumarate esters and analogous probes equipped with a click handle.
Figure 3
Figure 3. Mechanism of cysteine addition to allenamides. The prevalence of the mesomeric structure 9a rationalizes the formation of the nonconjugated product. An alternative mechanism involving attack of the neutral thiol to form a zwitterionic species followed by proton transfer was proposed by Loh and co-workers.
Figure 4
Figure 4. Osimertinib-derived allenamides as EGFR inhibitors.
Figure 5
Figure 5. Propiolonitriles as potential TCI warheads: (A) 3-Aryl and 3-alkyl propiolonitriles. (B) Mechanism of cysteine addition and thiol exchange.
Figure 6
Figure 6. 6-Ethynylthienopyrimidine as covalent ErbB kinase inhibitors. (A) 6-Ethynylthieno[3,2-d]pyrimidine and 6-ethynylthieno[2,3-d]pyrimidine-derived inhibitors. (B) Suggested mechanism of cysteine addition.
Figure 7
Figure 7. X-ray crystal structure of compound 17 in complex with the ErbB4 kinase domain (PDB 2R4B). The terminal alkyne moiety has reacted with Cys803 to form a vinyl thioether adduct. The N1-atom of the pyrimidine ring is further anchored to the backbone NH of Met799 in the hinge region via a hydrogen bond. The pyrimidine N3-atom is engaged in a water-mediated hydrogen bond to the side chain of Thr860 preceding the conserved DFG motif.
Figure 8
Figure 8. 2-Vinylpyrimidine-derived H4 receptor ligand VUF14480 and the unreactive analogue VUF14481.
Figure 9
Figure 9. Nonactivated terminal alkynes as cysteine traps. (A) Reaction of C-terminally propargylated ubiquitin 24 with the active site cysteine in DUBs. (B) Reactive (top) and nonreactive (bottom) analogues.
Figure 10
Figure 10. SNAr-based covalent ligands. (A) Classical mechanism of the SNAr reaction with cystein. (B) Selected examples of early SNAr-based ligands and reagents. Leaving groups are highlighted in red.
Figure 11
Figure 11. Covalent inhibitors antagonizing the interaction of ZAP-70 and Syk with ITAMs. Leaving groups are highlighted in red.
Figure 12
Figure 12. Electron-deficient (hetero)aryl probes used for evaluation of SNAr-based labeling in proteomes. (A) General structures. (B) Selected compounds preferably labeling cysteine or lysine.
Figure 13
Figure 13. 4-Halopyridines as quiescent SNAr electrophiles. (A) SNAr-reaction with 4-chloropyridine according to the classical mechanism. An anionic Meisenheimer intermediate is formed. (B) Analogous mechanism of the reaction with N-methyl-4-chloropyridine. A neutral dihydropyridine species is formed as the intermediate. (C) General structure of the investigated compounds. (D) Alkyne-tagged probes used for proteomic analysis.
Figure 14
Figure 14. (A) Structure of the covalent FGFR4 inhibitor 41. (B) X-ray cystal structure of 41 in complex with the FGFR4 kinase domain (PDB 5NUD). Both pyridine rings form a hydrogen bond with the backbone NH group of Ala553, while the nitro group stabilizes the active conformation via an intramolecular hydrogen bond with the diarylamino NH. The covalent bond with Cys552 is formed by SNAr displacement of the 6-chloro group from the 2-amino-3-nitropyridine moiety. A weak water mediated H-bond between the nitro substituent and the Arg483 guanidinium group was omitted for clarity.
Figure 15
Figure 15. EGFR inhibitors with SNAr warheads which do not form the predicted covalent bond with Cys797.
Figure 16
Figure 16. Development of covalent HCV NS5B polymerase inhibitors with SNAr warheads.
Figure 17
Figure 17. Second generation NS5B polymerase inhibitors with SNAr warheads. (A) Reversibly binding 2-chloropyridine 49 and the irreversibly binding quinoline analogue 50. (B) X-ray crystal structure of key compound 50 covalently bound to Cys366 of HCV NS5B polymerase (PDB 4MZ4). The compound is further anchored by hydrogen bonds between the 2-pyridone moiety and the backbone carbonyl atom of Gln446 and the NH group of Tyr448. The carboxylate and the quinoline N1-atom are linked to different residues via water-mediated hydrogen bond networks (the second sphere of water molecules and beyond was omitted for clarity). A second conformation of the Cys366 was omitted as well.
Figure 18
Figure 18. Strain-release reagents and their reaction with a cysteine-containing peptide.
Figure 19
Figure 19. Alkyl halides as CRGs (A) General mechanism of the SN2 reaction. (B) Dual attraction model rationalizing the enhanced reactivity of α-halocarbonyl compounds. (C) Reactivities of α-halopropion- and acetamides in a GSH assay. Half-lives were determined in the presence of 10 mM GSH at pH 7.4 and 37 °Ca or 60 °Cb. N-Phenylacrylamide is shown for comparison. (D) 2-Chloropropionamide (S)-53, a covalent PDIA1 inhibitor.
Figure 20
Figure 20. 5-Chloromethyl-1,2,3-triazoles as covalent MGMT inhibitors.
Figure 21
Figure 21. Examples of epoxide-containing drugs.
Figure 22
Figure 22. α-Acyl epoxides as warheads for putatively covalent EGFR inhibitors.
Figure 23
Figure 23. Ruxolitinib-derived triazoles with a propylene oxide warhead as selective JAK3 inhibitors.
Figure 24
Figure 24. K-Ras G12D or G12C-targeted covalent inhibitors. Key compound 66a features an aziridine warhead.
Figure 25
Figure 25. X-ray crystal structure of the K-Ras G12C mutant covalently bound to compound 66a (PDB 5V6V). Cys12 forms the covalent bond by opening the aziridine ring at the β-position. The indole NH and quinazoline N1-atom are involved in charge-assisted hydrogen bonds to the side chains of of Asp69 and Arg68, respectively, while the piperidine carboxamide oxygen interacts with the side chains of Tyr96 and Asp92 via water-bridged hydrogen bonds.
Figure 26
Figure 26. 3-Nitropropionate, propionate-3-nitronate, and the suggested reaction mechanism with ICL.
Figure 27
Figure 27. α-Cyanoacrylamide-derived covalent-reversible inhibitors.
Figure 28
Figure 28. Optimization of covalent-reversible FGFR4 inhibitors possessing an aldehyde warhead.
Figure 29
Figure 29. Reactivities of activated nitriles in a GSH-based assay. Half-lives (increasing from left to right) were determined in the presence of 10 mM GSH at pH 7.4 and 37 °C. Data for common acrylamides are provided for comparison.
Figure 30
Figure 30. Bicalutamide-derived antiandrogens with a putative covalent-reversible binding mode. (A) Chemical structures of the compounds studied by England and co-workers. (B) Suggested mechanism of covalent binding to Cys784 of the androgen receptor.
Figure 31
Figure 31. PF-303, a covalent-reversible BTK inhibitor featuring a cyanamide warhead.
Figure 32
Figure 32. Isothiocyanates as covalent-reversible warheads for cysteine and irreversible CRGs for lysine. (A) Common isothiocyanates found in cruciferous vegetables. (B) Reversible reaction of isothiocyanates with GSH or cysteines in proteins and slow thiourea formation, e.g., with lysine. Possible direct reaction pathways are depicted as dashed arrows.
Figure 33
Figure 33. Meisenheimer complex-forming electrophiles as putatively covalent-reversible PLK1 inhibitors. Mechanism and selected compounds.
Figure 34
Figure 34. Reversible cysteine targeting by disulfide bond formation. (A) Selected examples of headgroups known to generate disulfide bonds. (B) FAUC50, a covalent ligand which enabled crystallographic structure determination of the β2 adrenergic receptor.
Figure 35
Figure 35. Carbon acids adressing cysteine sulfenic acids. (A) Model system used by the Carroll group to assess sulfenic acid labeling with C-nuclophiles. A possible alternative reaction pathway is highlighted by the dashed arrow. (B) Cysteine sulfenic acid formation by ROS and trapping with dimedone. (C) Reactivities of cyclic C-nucleophiles. (D,E) Reactivities of linear C-nucleophiles. (F) Tofacitinib, an approved JAK inhibitor shown to react with sulfenic acids. Reactivity is expressed by pseudo-first-order rate constants derived from an LC-MS assay using the model system shown in (A).
Figure 36
Figure 37
Figure 37. Probes and warheads used to indentify ligandable lysines by chemical proteomics.
Figure 38
Figure 38. Reaction mechanism of wortmannin with Lys833 in PI3Kγ.
Figure 39
Figure 39. Reactivity of different Michael acceptors and activated nitriles toward lysine and GSH. Compounds with a preference for GSH are shown the upper row, while such preferably reacting with N-α-acetyl lysine are depicted in the lower row. Half-lives were determined in the presence of 50 mM N-α-acetyl lysine at pH 10.2 and 37 °C or 10 mM GSH at pH 7.4 and 37 °C.
Figure 40
Figure 40. Vinyl sulfone targeting a lysine in the solvent-exposed front region of CDK2. (A) Covalent and noncovalent inhibitors. (B) X-ray crystal structure of vinyl sulfone 98a covalently bound to Lys89 flanking the solvent-exposed front region of CDK2 (PDB 5CYI). The purine NH is hydrogen-bonded to the backbone carbonyl atom of Glu81, while the N3-atom and the diaryl NH are anchored to the backbone of Leu83 by two additional hydrogen bonds. Another water-bridged hydrogen bond links the purine N7-atom to the backbone NH group of Asp145 in the DFG motif. Further direct and water-mediated hydrogen bonds are established by the sulfonyl group. The N-terminal lobe was omitted for clarity.
Figure 41
Figure 41. Sulfur (VI) fluorides for lysine and tyrosine targeting.
Figure 42
Figure 42. Examples of sulfonyl fluorides applied in medicinal chemistry and chemical biology.
Figure 43
Figure 43. Compounds used for assessing the reactivity of sulfur (VI) fluorides toward different amino acids. (A) sulfonyl fluorides. (B) sulfonimidoyl fluorides. (C) aryl fluorosulfates. (D) Mechanism of the reaction between phenylsulfonyl fluoride and NAC.
Figure 44
Figure 44. Lysine-targeted m-5′-FSBA analogues for the evaluation of the relationship between warhead reactivity and FGFR1 inhibitory activity.
Figure 45
Figure 45. (A) Promiscuous kinase probe XO44. (B) X-ray crystal structure of XO44 covalently bound to the conserved Lys295 in the kinase SRC (PDB 5K9I). The 3-aminopyrazole is anchored to the hinge region by three hydrogen bonds involving the backbone of Glu339 and Met341. The sulfonyl group forms two additional hydrogen bonds with Phe278 and Gly279 in the glycine-rich loop, while the propargyl amide tag is oriented toward the bulk solvent without being involved in specific interactions.
Figure 46
Figure 46. Fluorosulfates for lysine targeting. (A) Aryl fluorosulfates and sulfonyl fluorides designed for adressing Lys15 in human transthyretin. (B) X-ray crystal structure of 113b bound to human transthyretin (PDB 4YDM). Unexpectedly, the free Lys15 ε-sulfamate was observed instead of the covalently bound ligand. The ligand is located in a relatively shallow pocket on the protein surface forming only a single conserved hydrogen bond between the hydroxy group of the dichlorophenol moiety and the side chain of Ser117. An alternative orientation of the 3-hydroxyphenyl residue was omitted for clarity.
Figure 47
Figure 47. Development of activated esters covalently targeting Lys779 in PI3Kδ.
Figure 48
Figure 48. N-Acyl-N-alkyl sulfonamides addressing surface-exposed lysine side chains. (A) Biotin-transferring probes. (B) Covalent ligand design and application example. The transferable residue is highlighted in red.
Figure 49
Figure 49. 2-Formylbenzenboronic acid reversibly forming stabilized Schiff bases with amines.
Figure 50
Figure 50. 2-Formylbenzenboronic acids and analogous acetophenones targeting MCL-1 via Schiff base formation.
Figure 51
Figure 51. Design of tyrosine-targeted sulfonyl fluorides as DcpS inhibitors.
Figure 52
Figure 52. Distinct binding modes of 124a–c in the respective X-ray crystal structures in complex with DcpS. The ligand is completely embedded in the protein environment, and the 2,4-diaminquinazoline core adopts a similar orientation in all structures. Conserved interactions include two hydrogen bonds of the quinazoline 2-amino group to the carboxylate of Glu185 and the backbone carbonyl of Pro204 as well as a hydrogen bond between the quinazoline 4-amino group and the Asp205 side chain. The second proton of the quinazoline 4-amine forms an intramolecular H-bond to the ether linker. The (protonated) quinazoline N1-atom is also hydrogen-bonded to Glu185. (A) ortho-Substituted derivative 124a covalently bound to Tyr113 (PDB 4QDE). The phenyl ring points to the “bottom” of the binding pocket. Additional hydrogen bonds are formed to the side chains of Lys142 and Tyr273. (B) meta-Substituted derivative 124b covalently bound to Tyr113 (PDB 4QEB). Covalent attachment is enabled by an “upward” orientation of the phenyl ring, giving rise to an additional hydrogen bond between the sulfonyl group and the Tyr143 side chain. (C) para-Substituted derivative 124c covalently bound to Tyr143 (PDB 4QDV). The overall orientation resembles that of 124a, but Tyr143 is labeled instead of Tyr113. No hydrogen bonds with Lys142, Tyr273 and His139 are observed in (B) and (C), and the latter two residues were omitted for clarity. No covalent modification of the proximal nucleophiles Lys142 and His139 was observed in any of the experiments.
Figure 53
Figure 53. Design of sulfonyl fluoride SRPKIN-1, the first tyrosine-targeted covalent kinase inhibitor.
Figure 54
Figure 54. Aryl fluorosulfate probes targeting CRABP2 used in chemical proteomics studies.
Figure 55
Figure 55. X-ray crystal structure of the fluorosulfate-based ligand 128 covalently bound to Tyr134 in CRABP2 (PDB 5HZQ). The ligand is deeply buried in the binding site, and the sulfate group is engaged in a direct hydrogen bond to the Arg132 side chain and water-mediated hydrogen bonds to the Arg111 side chain. The PEG-linker is not resolved and a second, slightly deviating conformation of the ligand and the Tyr134 side chain was omitted for clarity.
Figure 56
Figure 56. Alkyne-tagged aryl fluorosulfate-based probes used in an “inverse drug discovery” approach.
Figure 57
Figure 57. LAS17, a tyrosine-targeted dichlorotriazine-derived GSTP1 inhibitor.
Figure 58
Figure 58. (A) Aryl fluorosulfate-based inhibitor FS-p1 targeting Ser272 in DcpS. (B) Proposed mechanism for the formation of the dehydroalanine elimination product.
Figure 59
Figure 59. Glutamate-targeted PDE6δ inhibitors. (A) Attachment of N-methyl isoxazolium warheads to reversible inhibitors exemplified by 135. (B) Mechanism of the reaction between the N-methyl isoxazolium group and carboxylates.
Figure 60
Figure 60. X-ray crystal structure of compound 140d covalently bound to Glu88 of PDE6δ (PDB 5NAL). The ligand is predominantly bound in the less stable O-acylated form and deeply buried in the binding site. Hydrogen bonds are formed by both oxygen atoms of the first sulfonyl group to the side chains of Arg61 and Gln78. An additional hydrogen bond is established between the second sulfonyl group and the side chain of Tyr149 (omitted for clarity).
Figure 61
Figure 61. Spiro-epoxides as histidine-targeted covalent inhibitors of hMetAP2. (A) Former drug candidate beloranib. (B) Reaction of the natural product fumagillin with His231 in hMetAP2. (C) Simplified fumagillin-derived structures. (D) hMetAP2 inhibitor 145a with improved PK properties.
Figure 62
Figure 62. X-ray crystal structure of the covalent complex between hMetAP2 and compound 145a (PDB 5CLS). His231 is covalently attached to the methylene group formed from the terminal carbon atom of the epoxide ring. The ensuing hydroxy group is linked to Asp251 and His382 by water-mediated hydrogen bonds. A direct hydrogen bond between the carbamate’s carbonyl group and the Asn329 backbone NH, and further water-mediated hydrogen bonds additionally anchor the ligand in the binding site.
Figure 63
Figure 63. α-Cyanoenones as histidine-targeted covalent-reversible IDH1 inhibitors. (A) Hit compound 146a and optimized derivatives. (B) X-ray crystal structure of 146c bound to IDH1 (PDB 6BL1). His315 is covalently attached to the β-position of the enone precursor. A key hydrogen bond is formed between the α-cyano moiety and the backbone NH of Se326. The enone keto group is hydrogen-bonded to the Lys374 side chain, while the diarylamino group forms a charge-assisted hydrogen bond to the carboxylate of Asp375.
Figure 64
Figure 64. Methionine-targeted oxaziridines. (A) Urea and carbamate-derived analogues. (B) Reaction with methionine via S-imidation or concomitant S-oxidation. Conditions: (1) D2O/CD3OD = 1:1, 2.5 min or (2) D2O/CD3OD = 95:5, 20 min. (C) Reaction mechanism of covalent methionine modification.
References
ARTICLE SECTIONSThis article references 351 other publications.
- 1Mann, M.; Jensen, O. N. Proteomic Analysis of Post-Translational Modifications. Nat. Biotechnol. 2003, 21 (3), 255– 261, DOI: 10.1038/nbt0303-255[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhsFajsro%253D&md5=ea9706baed997144456697914d190260Proteomic analysis of post-translational modificationsMann, Matthias; Jensen, Ole N.Nature Biotechnology (2003), 21 (3), 255-261CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)A review. Post-translational modifications modulate the activity of most eukaryote proteins. Anal. of these modifications presents formidable challenges but their detn. generates indispensable insight into biol. function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purifn. of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in mol. detail, novel mass spectrometric peptide sequencing and anal. technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.
- 2Schopfer, F. J.; Cipollina, C.; Freeman, B. A. Formation and Signaling Actions of Electrophilic Lipids. Chem. Rev. 2011, 111 (10), 5997– 6021, DOI: 10.1021/cr200131e[ACS Full Text], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1SltrvI&md5=3fc9a6a520d8ca4dfc2ff3ad8545c160Formation and Signaling Actions of Electrophilic LipidsSchopfer, Francisco J.; Cipollina, Chiara; Freeman, Bruce A.Chemical Reviews (Washington, DC, United States) (2011), 111 (10), 5997-6021CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. This review addresses lipid-derived electrophilic species by first focusing on biochem. characteristics of electrophiles and the mechanisms underlying the endogenous formation of electrophilic species. This information is placed in the context of the cell signaling pathways modulated by electrophilic fatty acid derivs. and how these reactions can impact physiol. and pathophysiol. processes.
- 3Allis, C. D.; Jenuwein, T. The Molecular Hallmarks of Epigenetic Control. Nat. Rev. Genet. 2016, 17 (8), 487– 500, DOI: 10.1038/nrg.2016.59[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGiur%252FJ&md5=dbbff4b5de8a0316f16664f73f3ad16dThe molecular hallmarks of epigenetic controlAllis, C. David; Jenuwein, ThomasNature Reviews Genetics (2016), 17 (8), 487-500CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)Over the past 20 years, breakthrough discoveries of chromatin-modifying enzymes and assocd. mechanisms that alter chromatin in response to physiol. or pathol. signals have transformed our knowledge of epigenetics from a collection of curious biol. phenomena to a functionally dissected research field. Here, we provide a personal perspective on the development of epigenetics, from its historical origins to what we define as 'the modern era of epigenetic research'. We primarily highlight key mol. mechanisms of and conceptual advances in epigenetic control that have changed our understanding of normal and perturbed development.
- 4Uetrecht, J. Idiosyncratic Drug Reactions: Current Understanding. Annu. Rev. Pharmacol. Toxicol. 2007, 47 (1), 513– 539, DOI: 10.1146/annurev.pharmtox.47.120505.105150[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXit1altb0%253D&md5=587b03e88f3b881c87d383818bea7f47Idiosyncratic drug reactions: current understandingUetrecht, JackAnnual Review of Pharmacology and Toxicology (2007), 47 (), 513-539CODEN: ARPTDI; ISSN:0362-1642. (Annual Reviews Inc.)A review. Clin. characteristics and circumstantial evidence suggest that idiosyncratic drug reactions are caused by reactive metabolites and are immune-mediated; however, there are few definitive data and there are likely exceptions. There are three principal hypotheses for how reactive metabolites might induce an immune-mediated idiosyncratic reaction: the hapten hypothesis, the danger hypothesis, and the PI hypothesis. It has been proposed that some idiosyncratic reactions, esp. those involving the liver, represent metabolic idiosyncrasy; however, there are even less data to support this hypothesis. The unpredictable nature of these reactions makes mechanistic studies difficult. There is a very strong assocn. with specific human leukocyte antigen (HLA) genes for certain reactions, but this has only been demonstrated for very few drugs. Animal models represent a very powerful tool for mechanistic studies, but the no. of valid models is also limited. There may be biomarkers of risk; however, much more work needs to be done.
- 5Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The Resurgence of Covalent Drugs. Nat. Rev. Drug Discovery 2011, 10 (4), 307– 317, DOI: 10.1038/nrd3410[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktVGmu7g%253D&md5=2190289081e151416c097be4a5b04460The resurgence of covalent drugsSingh, Juswinder; Petter, Russell C.; Baillie, Thomas A.; Whitty, AdrianNature Reviews Drug Discovery (2011), 10 (4), 307-317CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Covalent drugs haveproved to be successful therapies for various indications, but largely owing to safety concerns, they are rarely considered when initiating a target-directed drug discovery project. There is a need to reassess this important class of drugs, and to reconcile the discordance between the historic success of covalent drugs and the reluctance of most drug discovery teams to include them in their armamentarium. This Review surveys the prevalence and pharmacol. advantages of covalent drugs, discusses how potential risks and challenges may be addressed through innovative design, and presents the broad opportunities provided by targeted covalent inhibitors.
- 6Bauer, R. A. Covalent Inhibitors in Drug Discovery: From Accidental Discoveries to Avoided Liabilities and Designed Therapies. Drug Discovery Today 2015, 20 (9), 1061– 1073, DOI: 10.1016/j.drudis.2015.05.005[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptlOnu7s%253D&md5=6ba5b016d2804c7d56838a2724b9a0b9Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapiesBauer, Renato A.Drug Discovery Today (2015), 20 (9), 1061-1073CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)Drugs that covalently bond to their biol. targets have a long history in drug discovery. A look at drug approvals in recent years suggests that covalent drugs will continue to make impacts on human health for years to come. Although fraught with concerns about toxicity, the high potencies and prolonged effects achievable with covalent drugs may result in less-frequent drug dosing and in wide therapeutic margins for patients. Covalent inhibition can also dissoc. drug pharmacodynamics (PD) from pharmacokinetics (PK), which can result in desired drug efficacy for inhibitors that have short systemic exposure. Evidence suggests that there is a reduced risk for the development of resistance against covalent drugs, which is a major challenge in areas such as oncol. and infectious disease.
- 7Powers, J. C.; Asgian, J. L.; Ekici, Ö. D.; James, K. E. Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases. Chem. Rev. 2002, 102 (12), 4639– 4750, DOI: 10.1021/cr010182v[ACS Full Text], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XosFSnsr4%253D&md5=eb21ff0297b87537a7d459c92f13c75dIrreversible inhibitors of serine, cysteine, and threonine proteasesPowers, James C.; Asgian, Juliana L.; Ekici, Oezlem Dogan; James, Karen EllisChemical Reviews (Washington, DC, United States) (2002), 102 (12), 4639-4750CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Topics discussed include alkylating, acylating, phosphonylating, and sulfonylating agents.
- 8Bachovchin, D. A.; Cravatt, B. F. The Pharmacological Landscape and Therapeutic Potential of Serine Hydrolases. Nat. Rev. Drug Discovery 2012, 11 (1), 52– 68, DOI: 10.1038/nrd3620[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFOktA%253D%253D&md5=4fde1e23117ad11848a9c2eef85376b7The pharmacological landscape and therapeutic potential of serine hydrolasesBachovchin, Daniel A.; Cravatt, Benjamin F.Nature Reviews Drug Discovery (2012), 11 (1), 52-68CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Serine hydrolases perform crucial roles in many biol. processes, and several of these enzymes are targets of approved drugs for indications such as type 2 diabetes, Alzheimer's disease and infectious diseases. Despite this, most of the human serine hydrolases (of which there are more than 200) remain poorly characterized with respect to their physiol. substrates and functions, and the vast majority lack selective, in vivo-active inhibitors. Here, we review the current state of pharmacol. for mammalian serine hydrolases, including marketed drugs, compds. that are under clin. investigation and selective inhibitors emerging from academic probe development efforts. We also highlight recent methodol. advances that have accelerated the rate of inhibitor discovery and optimization for serine hydrolases, which we anticipate will aid in their biol. characterization and, in some cases, therapeutic validation.
- 9Paul, S. M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.; Lindborg, S. R.; Schacht, A. L. How to Improve R&D Productivity: The Pharmaceutical Industry’s Grand Challenge. Nat. Rev. Drug Discovery 2010, 9 (3), 203– 214, DOI: 10.1038/nrd3078[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitFemsbg%253D&md5=2f32bcc48c869290eef18ff9400afcc5How to improve R&D productivity: the pharmaceutical industry's grand challengePaul, Steven M.; Mytelka, Daniel S.; Dunwiddie, Christopher T.; Persinger, Charles C.; Munos, Bernard H.; Lindborg, Stacy R.; Schacht, Aaron L.Nature Reviews Drug Discovery (2010), 9 (3), 203-214CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. The pharmaceutical industry is under growing pressure from a range of environmental issues, including major losses of revenue owing to patent expirations, increasingly cost-constrained healthcare systems and more demanding regulatory requirements. In our view, the key to tackling the challenges such issues pose to both the future viability of the pharmaceutical industry and advances in healthcare is to substantially increase the no. and quality of innovative, cost-effective new medicines, without incurring unsustainable R&D costs. However, it is widely acknowledged that trends in industry R&D productivity have been moving in the opposite direction for a no. of years. Here, we present a detailed anal. based on comprehensive, recent, industry-wide data to identify the relative contributions of each of the steps in the drug discovery and development process to overall R&D productivity. We then propose specific strategies that could have the most substantial impact in improving R&D productivity.
- 10Bandyopadhyay, A.; Gao, J. Targeting Biomolecules with Reversible Covalent Chemistry. Curr. Opin. Chem. Biol. 2016, 34, 110– 116, DOI: 10.1016/j.cbpa.2016.08.011[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVOqtrrE&md5=1294e2befed2127ceec6a7e7ae0dd2f3Targeting biomolecules with reversible covalent chemistryBandyopadhyay, Anupam; Gao, JianminCurrent Opinion in Chemical Biology (2016), 34 (), 110-116CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)Interaction of biomols. typically proceeds in a highly selective and reversible manner, for which covalent bond formation has been largely avoided due to the potential difficulty of dissocn. However, employing reversible covalent warheads in drug design has given rise to covalent enzyme inhibitors that serve as powerful therapeutics, as well as mol. probes with exquisite target selectivity. This review article summarizes the recent advances in the development of reversible covalent chem. for biol. and medicinal applications. Specifically, we document the chem. strategies that allow for reversible modification of the three major classes of nucleophiles in biol.: thiols, alcs. and amines. Emphasis is given to the chem. mechanisms that underlie the development of these reversible covalent reactions and their utilization in biol.
- 11Bradshaw, J. M.; McFarland, J. M.; Paavilainen, V. O.; Bisconte, A.; Tam, D.; Phan, V. T.; Romanov, S.; Finkle, D.; Shu, J.; Patel, V.; Ton, T.; Li, X.; Loughhead, D. G.; Nunn, P. A.; Karr, D. E.; Gerritsen, M. E.; Funk, J. O.; Owens, T. D.; Verner, E.; Brameld, K. A.; Hill, R. J.; Goldstein, D. M.; Taunton, J. Prolonged and Tunable Residence Time Using Reversible Covalent Kinase Inhibitors. Nat. Chem. Biol. 2015, 11 (7), 525– 531, DOI: 10.1038/nchembio.1817[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFeju7%252FE&md5=85a73d9ecd62695d03166f7009c68dd8Prolonged and tunable residence time using reversible covalent kinase inhibitorsBradshaw, J. Michael; McFarland, Jesse M.; Paavilainen, Ville O.; Bisconte, Angelina; Tam, Danny; Phan, Vernon T.; Romanov, Sergei; Finkle, David; Shu, Jin; Patel, Vaishali; Ton, Tony; Li, Xiaoyan; Loughhead, David G.; Nunn, Philip A.; Karr, Dane E.; Gerritsen, Mary E.; Funk, Jens Oliver; Owens, Timothy D.; Verner, Erik; Brameld, Ken A.; Hill, Ronald J.; Goldstein, David M.; Taunton, JackNature Chemical Biology (2015), 11 (7), 525-531CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here the authors made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, the authors identified potent and selective BTK inhibitors that demonstrated biochem. residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.
- 12Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. J. Med. Chem. 2014, 57 (23), 10072– 10079, DOI: 10.1021/jm501412a[ACS Full Text], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVyjtLbM&md5=3ccf59ab7a494655185e5eb5becf8c48Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible InhibitorsFlanagan, Mark E.; Abramite, Joseph A.; Anderson, Dennis P.; Aulabaugh, Ann; Dahal, Upendra P.; Gilbert, Adam M.; Li, Chao; Montgomery, Justin; Oppenheimer, Stacey R.; Ryder, Tim; Schuff, Brandon P.; Uccello, Daniel P.; Walker, Gregory S.; Wu, Yan; Brown, Matthew F.; Chen, Jinshan M.; Hayward, Matthew M.; Noe, Mark C.; Obach, R. Scott; Philippe, Laurence; Shanmugasundaram, Veerabahu; Shapiro, Michael J.; Starr, Jeremy; Stroh, Justin; Che, YeJournal of Medicinal Chemistry (2014), 57 (23), 10072-10079CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry addnl. risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biol. relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.
- 13Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.; Horning, B. D.; González-Páez, G. E.; Chatterjee, S.; Lanning, B. R.; Teijaro, J. R.; Olson, A. J.; Wolan, D. W.; Cravatt, B. F. Proteome-Wide Covalent Ligand Discovery in Native Biological Systems. Nature 2016, 534 (7608), 570– 574, DOI: 10.1038/nature18002[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVSksbnN&md5=6fab087c042de735f8a5931e9f5b946cProteome-wide covalent ligand discovery in native biological systemsBackus, Keriann M.; Correia, Bruno E.; Lum, Kenneth M.; Forli, Stefano; Horning, Benjamin D.; Gonzalez-Paez, Gonzalo E.; Chatterjee, Sandip; Lanning, Bryan R.; Teijaro, John R.; Olson, Arthur J.; Wolan, Dennis W.; Cravatt, Benjamin F.Nature (London, United Kingdom) (2016), 534 (7608), 570-574CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Small mols. are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-mol. ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-mol. probes for proteins that have proven difficult to target using high-throughput screening of complex compd. libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-mol. probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quant. anal. of cysteine-reactive small-mol. fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chem. probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compds. that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines vs. primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compds. that can illuminate protein functions in native biol. systems.
- 14Miller, R. M.; Paavilainen, V. O.; Krishnan, S.; Serafimova, I. M.; Taunton, J. Electrophilic Fragment-Based Design of Reversible Covalent Kinase Inhibitors. J. Am. Chem. Soc. 2013, 135 (14), 5298– 5301, DOI: 10.1021/ja401221b[ACS Full Text], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltVClu7g%253D&md5=c6100b56ce6f587f6afb834aeafec351Electrophilic Fragment-Based Design of Reversible Covalent Kinase InhibitorsMiller, Rand M.; Paavilainen, Ville O.; Krishnan, Shyam; Serafimova, Iana M.; Taunton, JackJournal of the American Chemical Society (2013), 135 (14), 5298-5301CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Fragment-based ligand design and covalent targeting of noncatalytic cysteines have been employed to develop potent and selective kinase inhibitors. Here, we combine these approaches, starting with a panel of low-mol.-wt., heteroaryl-substituted cyanoacrylamides, which we have previously shown to form reversible covalent bonds with cysteine thiols. Using this strategy, we identify electrophilic fragments with sufficient ligand efficiency and selectivity to serve as starting points for the first reported inhibitors of the MSK1 C-terminal kinase domain. Guided by X-ray co-crystal structures, indazole fragment 1 was elaborated to afford 12 (RMM-46), a reversible covalent inhibitor that exhibits high ligand efficiency and selectivity for MSK/RSK-family kinases. At nanomolar concns., 12 blocked activation of cellular MSK and RSK, as well as downstream phosphorylation of the crit. transcription factor, CREB.
- 15Jöst, C.; Nitsche, C.; Scholz, T.; Roux, L.; Klein, C. D. Promiscuity and Selectivity in Covalent Enzyme Inhibition: A Systematic Study of Electrophilic Fragments. J. Med. Chem. 2014, 57 (18), 7590– 7599, DOI: 10.1021/jm5006918[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M%252FktlKisA%253D%253D&md5=d89c64d854194704eb068147961de4afPromiscuity and selectivity in covalent enzyme inhibition: a systematic study of electrophilic fragmentsJost Christian; Nitsche Christoph; Scholz Therese; Roux Lionel; Klein Christian DJournal of medicinal chemistry (2014), 57 (18), 7590-9 ISSN:.Covalent ligand-target interactions offer significant pharmacological advantages. However, off-target reactivity of the reactive groups, which usually have electrophilic properties, must be minimized, and the selectivity of irreversible inhibitors is a crucial requirement. We therefore performed a systematic study to determine the selectivity of several electrophilic groups that can be used as building blocks for covalently binding ligands. Six reactive groups with modulated electrophilicity were combined with 11 nonreactive moieties, resulting in a small combinatorial library of 72 fragment-like compounds. These compounds were screened against a group of 11 enzyme targets to assess their selectivity and their potential for promiscuous binding to proteins. The assay results showed a considerably lower degree of promiscuity than initially expected, even for those members of the screening collection that contain supposedly highly reactive electrophiles.
- 16Kathman, S. G.; Xu, Z.; Statsyuk, A. V. A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases. J. Med. Chem. 2014, 57 (11), 4969– 4974, DOI: 10.1021/jm500345q[ACS Full Text], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXovVWiur0%253D&md5=39dee964ac1ba26ed373eedbd86102acA Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine ProteasesKathman, Stefan G.; Xu, Ziyang; Statsyuk, Alexander V.Journal of Medicinal Chemistry (2014), 57 (11), 4969-4974CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A novel fragment-based drug discovery approach is reported which irreversibly tethers drug-like fragments to catalytic cysteines. Acrylamides, acylaminoacrylates, vinylsulfonamides, and acylaminopropenyl sulfones were prepd. and the dependence of their rates of reaction with N-acetylcysteine Me ester on their structure was detd.; of the compds. tested, the rate of Michael addn. of N-acetylcysteine Me ester to acylaminoacrylates depended least on the acyl moiety. A library of 100 fragment-substituted acylaminoacrylates was prepd.; addn. of subsets of the library to papain followed by mass spectrometric anal. identified three acylaminoacrylates which selectively reacted with the cysteine protease papain. The kinetics of the inhibition of papain by the acylaminoacrylates, the effect of known inhibitors of papain on its inhibition by the acylaminoacrylates, and the lack of inhibition of other cysteine proteases (human rhinovirus 3C protease, the catalytic domain of the deubiquitinase USP08, and the E2 ubiquitin-conjugating enzyme UbcH7) by the acylaminoacrylates supported their identification as selective and irreversible papain inhibitors.
- 17Ostrem, J. M.; Peters, U.; Sos, M. L.; Wells, J. A.; Shokat, K. M. K-Ras(G12C) Inhibitors Allosterically Control GTP Affinity and Effector Interactions. Nature 2013, 503 (7477), 548– 551, DOI: 10.1038/nature12796[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVGqs7jO&md5=27c1e7d8ef6b7e4b2919e9f00a073923K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactionsOstrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.Nature (London, United Kingdom) (2013), 503 (7477), 548-551CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally assocd. with poor response to std. therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concn. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small mols. that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compds. rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallog. studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favor GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.
- 18Zimmermann, G.; Rieder, U.; Bajic, D.; Vanetti, S.; Chaikuad, A.; Knapp, S.; Scheuermann, J.; Mattarella, M.; Neri, D. A Specific and Covalent JNK-1 Ligand Selected from an Encoded Self-Assembling Chemical Library. Chem. - Eur. J. 2017, 23 (34), 8152– 8155, DOI: 10.1002/chem.201701644[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXos1eitbY%253D&md5=5109df70628c1a2178cab73d645d82c9A Specific and Covalent JNK-1 Ligand Selected from an Encoded Self-Assembling Chemical LibraryZimmermann, Gunther; Rieder, Ulrike; Bajic, Davor; Vanetti, Sara; Chaikuad, Apirat; Knapp, Stefan; Scheuermann, Joerg; Mattarella, Martin; Neri, DarioChemistry - A European Journal (2017), 23 (34), 8152-8155CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)We describe the construction of a DNA-encoded chem. library comprising 148 135 members, generated through the self-assembly of two sub-libraries, contg. 265 and 559 members, resp. The library was designed to contain building blocks potentially capable of forming covalent interactions with target proteins. Selections performed with JNK1, a kinase contg. a conserved cysteine residue close to the ATP binding site, revealed the preferential enrichment of a 2-phenoxynicotinic acid moiety (building block A82) and a 4-(3,4-difluorophenyl)-4-oxobut-2-enoic acid moiety (building block B272). When the two compds. were joined by a short PEG linker, the resulting bidentate binder (A82-L-B272) was able to covalently modify JNK1 in the presence of a large molar excess of glutathione (0.5 mM), used to simulate intracellular reducing conditions. By contrast, derivs. of the individual building blocks were not able to covalently modify JNK1 in the same exptl. conditions. The A82-L-B272 ligand was selective over related kinases (BTK and GAK), which also contain targetable cysteine residues in the vicinity of the active site.
- 19Zambaldo, C.; Daguer, J.-P.; Saarbach, J.; Barluenga, S.; Winssinger, N. Screening for Covalent Inhibitors Using DNA-Display of Small Molecule Libraries Functionalized with Cysteine Reactive Moieties. MedChemComm 2016, 7 (7), 1340– 1351, DOI: 10.1039/C6MD00242K[Crossref], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVCis77I&md5=799993f7f231acd4a9951106d1f9de56Screening for covalent inhibitors using DNA-display of small molecule libraries functionalized with cysteine reactive moietiesZambaldo, C.; Daguer, J.-P.; Saarbach, J.; Barluenga, S.; Winssinger, N.MedChemComm (2016), 7 (7), 1340-1351CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)DNA-encoded chem. libraries are increasingly used to identify leads for drug discovery or chem. biol. Despite the resurging interest in covalent inhibitors, libraries are typically designed with synthon filtered out for reactive functionalities that can engage a target through covalent interactions. Herein, we report the synthesis of two libraries contg. Michael acceptors to identify cysteine reactive ligands. We developed a simple procedure to discriminate between covalent and high affinity non-covalent inhibitors using DNA display of the library in a microarray format. The methodol. was validated with known covalent and high affinity non-covalent kinase inhibitors. Screening of the library revealed novel covalent inhibitors for MEK2 and ERBB2.
- 20Strelow, J. M. A Perspective on the Kinetics of Covalent and Irreversible Inhibition. SLAS Discov. 2017, 22 (1), 3– 20, DOI: 10.1177/1087057116671509[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlSntrbJ&md5=bd404e6b26df20b8761cd38f0f3c3541A perspective on the kinetics of covalent and irreversible inhibitionStrelow, John M.SLAS Discovery (2017), 22 (1), 3-20CODEN: SDLIBT; ISSN:2472-5560. (Sage Publications)The clin. and com. success of covalent drugs has prompted a renewed and more deliberate pursuit of covalent and irreversible mechanisms within drug discovery. A covalent mechanism can produce potent inhibition in a biochem., cellular, or in vivo setting. In many cases, teams choose to focus on the consequences of the covalent event, defined by an IC50 value. In a biochem. assay, the IC50 may simply reflect the target protein concn. in the assay. What has received less attention is the importance of the rate of covalent modification, defined by kinact/KI. The kinact/KI is a rate const. describing the efficiency of covalent bond formation resulting from the potency (KI) of the first reversible binding event and the max. potential rate (kinact) of inactivation. In this perspective, it is proposed that the kinact/KI should be employed as a crit. parameter to identify covalent inhibitors, interpret structure-activity relationships (SARs), translate activity from biochem. assays to the cell, and more accurately define selectivity. It is also proposed that a physiol. relevant kinact/KI and an (unbound) AUC generated from a pharmacokinetic profile reflecting direct exposure of the inhibitor to the target protein are two crit. determinants of in vivo covalent occupancy. A simple equation is presented to define this relationship and improve the interpretation of covalent and irreversible kinetics.
- 21Miyahisa, I.; Sameshima, T.; Hixon, M. S. Rapid Determination of the Specificity Constant of Irreversible Inhibitors (Kinact/Ki) by Means of an Endpoint Competition Assay. Angew. Chem., Int. Ed. 2015, 54 (47), 14099– 14102, DOI: 10.1002/anie.201505800[Crossref], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsF2ktr3N&md5=9491844cd2fde8dda986189aff7800d8Rapid Determination of the Specificity Constant of Irreversible Inhibitors (kinact/KI) by Means of an Endpoint Competition AssayMiyahisa, Ikuo; Sameshima, Tomoya; Hixon, Mark S.Angewandte Chemie, International Edition (2015), 54 (47), 14099-14102CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Owing to their covalent target occupancy, irreversible inhibitors require low exposures and offer long duration, and their use thus represents a powerful strategy for achieving pharmacol. efficacy. Importantly, the potency metric of irreversible inhibitors is kinact/KI not IC50. A simple approach to measuring kinact/KI was developed that makes use of an irreversible probe for competitive assays run to completion against test compds. In this system, the kinact/KI value of the test compd. is equal to (kinact/KI)probe×[probe]/IC50. The advantages of this method include simplicity, high throughput, and application to all target classes, and it only requires an in-depth kinetic evaluation of the probe.
- 22Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry. Annu. Rev. Biochem. 2008, 77 (1), 383– 414, DOI: 10.1146/annurev.biochem.75.101304.124125[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXos1ekur0%253D&md5=2f2c471169d2473f7024eeb41d4eaecdActivity-based protein profiling: From enzyme chemistry to proteomic chemistryCravatt, Benjamin F.; Wright, Aaron T.; Kozarich, John W.Annual Review of Biochemistry (2008), 77 (), 383-414CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chem. proteomic strategy to characterize enzyme function directly in native biol. systems on a global scale. Here, the authors review the basic technol. of ABPP, the enzyme classes addressable by this method, and the biol. discoveries attributable to its application.
- 23Lanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.; Hett, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.; Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.; Whiteley, L. O.; Hayward, M. M.; Cravatt, B. F. A Road Map to Evaluate the Proteome-Wide Selectivity of Covalent Kinase Inhibitors. Nat. Chem. Biol. 2014, 10 (9), 760– 767, DOI: 10.1038/nchembio.1582[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFygs7nO&md5=6f17b0d1b00e088f37c511eea577d290A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitorsLanning, Bryan R.; Whitby, Landon R.; Dix, Melissa M.; Douhan, John; Gilbert, Adam M.; Hett, Erik C.; Johnson, Theodore O.; Joslyn, Chris; Kath, John C.; Niessen, Sherry; Roberts, Lee R.; Schnute, Mark E.; Wang, Chu; Hulce, Jonathan J.; Wei, Baoxian; Whiteley, Laurence O.; Hayward, Matthew M.; Cravatt, Benjamin F.Nature Chemical Biology (2014), 10 (9), 760-767CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quant. MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an exptl. road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.
- 24Zaro, B. W.; Whitby, L. R.; Lum, K. M.; Cravatt, B. F. Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors. J. Am. Chem. Soc. 2016, 138 (49), 15841– 15844, DOI: 10.1021/jacs.6b10589[ACS Full Text], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFyrtbbI&md5=830d336c87c6efa70659545c46c87b4eMetabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible InhibitorsZaro, Balyn W.; Whitby, Landon R.; Lum, Kenneth M.; Cravatt, Benjamin F.Journal of the American Chemical Society (2016), 138 (49), 15841-15844CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrophilic small mols. are an important class of chem. probes and drugs that produce biol. effects by irreversibly modifying proteins. Examples of electrophilic drugs include covalent kinase inhibitors that are used to treat cancer and the multiple sclerosis drug di-Me fumarate. Optimized covalent drugs typically inactivate their protein targets rapidly in cells, but ensuing time-dependent, off-target protein modification can erode selectivity and diminish the utility of reactive small mols. as chem. probes and therapeutics. Here, the authors describe an approach to confer kinetic selectivity to electrophilic drugs. The authors show that an analog of the covalent Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib bearing a fumarate ester electrophile is vulnerable to enzymic metab. on a time-scale that preserves rapid and sustained BTK inhibition, while thwarting more slowly accumulating off-target reactivity in cell and animal models. These findings demonstrate that metabolically labile electrophilic groups can endow covalent drugs with kinetic selectivity to enable perturbation of proteins and biochem. pathways with greater precision.
- 25Serafimova, I. M.; Pufall, M. A.; Krishnan, S.; Duda, K.; Cohen, M. S.; Maglathlin, R. L.; McFarland, J. M.; Miller, R. M.; Frödin, M.; Taunton, J. Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles. Nat. Chem. Biol. 2012, 8 (5), 471– 476, DOI: 10.1038/nchembio.925[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvVKitrw%253D&md5=6455a9a029714beb317286f71ac1326bReversible targeting of noncatalytic cysteines with chemically tuned electrophilesSerafimova, Iana M.; Pufall, Miles A.; Krishnan, Shyam; Duda, Katarzyna; Cohen, Michael S.; Maglathlin, Rebecca L.; McFarland, Jesse M.; Miller, Rand M.; Froedin, Morten; Taunton, JackNature Chemical Biology (2012), 8 (5), 471-476CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacol. potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissocg., covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chem.-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.
- 26Noe, M. C.; Gilbert, A. M. Targeted Covalent Enzyme Inhibitors. In Annual Reports in Medicinal Chemistry; Desai, M. C., Ed.; Academic Press, 2012; Vol. 47, pp. 413– 439. DOI: 10.1016/B978-0-12-396492-2.00027-8 .
- 27Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing Irreversible Inhibitors of the Protein Kinase Cysteinome. Chem. Biol. 2013, 20 (2), 146– 159, DOI: 10.1016/j.chembiol.2012.12.006[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtFWitbs%253D&md5=cef80823123bae88362e816a0a638133Developing Irreversible Inhibitors of the Protein Kinase CysteinomeLiu, Qingsong; Sabnis, Yogesh; Zhao, Zheng; Zhang, Tinghu; Buhrlage, Sara J.; Jones, Lyn H.; Gray, Nathanael S.Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 146-159CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)A review. Protein kinases are a large family of approx. 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues, such as tyrosine, serine, and threonine, that serves as a ubiquitous mechanism for cellular signal transduction. The clin. success of a no. of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large no. of kinases may represent therapeutically relevant targets. To date, the majority of clin. and preclin. kinase inhibitors are ATP competitive, noncovalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently, there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a no. of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency, and ability to validate pharmacol. specificity through mutation of the reactive cysteine residue. Here, we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biol. activity profiles. In addn., we provided an informatics assessment of the potential "kinase cysteinome" and discuss strategies for the efficient development of new covalent inhibitors.
- 28Miller, R. M.; Taunton, J. Targeting Protein Kinases with Selective and Semipromiscuous Covalent Inhibitors. In Methods in Enzymology; Shokat, K. M., Ed.; Academic Press, 2014; Vol. 548, pp 93– 116. DOI: 10.1016/B978-0-12-397918-6.00004-5 .
- 29Gilbert, A. M. Recent Advances in Irreversible Kinase Inhibitors. Pharm. Pat. Anal. 2014, 3 (4), 375– 386, DOI: 10.4155/ppa.14.24[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslSkurnN&md5=e0d3394e575a3fdb4e16d4627b83baa5Recent advances in irreversible kinase inhibitorsGilbert, Adam M.Pharmaceutical Patent Analyst (2014), 3 (4), 375-386CODEN: PPAHCN; ISSN:2046-8954. (Future Science Ltd.)Despite concerns of off-target selectivity and cytotoxicity, there has been a resurgence in interest in irreversible kinase inhibitors resulting in more than 60 disclosed patent and patent applications over the past 4 years. Many of these inhibitors possess several key advantages over their reversible counterparts. The patent literature from 2010 to 2013 has been reviewed and novel irreversible kinase inhibitors for Bruton's tyrosine kinase, epidermal growth factor receptor, Janus kinase 3, phosphoinsitide 3 and other kinases are disclosed and discussed. These inhibitors offer novel treatments for mantle cell lymphoma, non-small-cell lung cancer, autoimmune disorders and severe metastatic cancers. A future perspective is presented on the likelihood of clin. success of these agents as well as the potential for new uses of irreversible kinase inhibitors in the future.
- 30Adeniyi, A. A.; Muthusamy, R.; Soliman, M. E. New Drug Design with Covalent Modifiers. Expert Opin. Drug Discovery 2016, 11 (1), 79– 90, DOI: 10.1517/17460441.2016.1115478[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht12qtL8%253D&md5=9307f11db2da4d9853b49a97cec7e0e2New drug design with covalent modifiersAdeniyi, Adebayo A.; Muthusamy, Ramesh; Soliman, Mahmoud ESExpert Opinion on Drug Discovery (2016), 11 (1), 79-90CODEN: EODDBX; ISSN:1746-0441. (Taylor & Francis Ltd.)Introduction: A major challenge for drug design is the alarming increase in drug resistance, mutations and toxicity. In recent years, covalent drugs have become a promising option to address these problems, due to their significant advantages. These advantages include their ability to target rare, non-conserved residues, shallow binding sites of target proteins and their ability to retain their binding with a receptor for a very long time. Areas covered: This review shows the increasing progress in rational design and virtual screening of covalent drugs and the promising future of accurately predicting effective covalent drugs through in silico screening. Expert opinion: All the current clin. approved covalent drugs were discovered by chance instead of systematic design. There is a promising and commendable effort towards high-throughput screening and the accurate discovery of new covalent inhibitors, which may address the problems of drug resistance and mutation. However, despite the current progression, there is still a need for more rational attention to improve the covalent warhead for improved receptor interaction and selectivity.
- 31Baillie, T. A. Targeted Covalent Inhibitors for Drug Design. Angew. Chem., Int. Ed. 2016, 55 (43), 13408– 13421, DOI: 10.1002/anie.201601091[Crossref], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlOrtb%252FN&md5=18bed2ae08dcb4245a42b3070abc6354Targeted Covalent Inhibitors for Drug DesignBaillie, Thomas A.Angewandte Chemie, International Edition (2016), 55 (43), 13408-13421CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)In contrast to the traditional mechanism of drug action that relies on the reversible, noncovalent interaction of a ligand with its biol. target, a targeted covalent inhibitor (TCI) is designed such that the initial, reversible assocn. is followed by the formation of a covalent bond between an electrophile on the ligand and a nucleophilic center in the protein. Although this approach offers a variety of potential benefits (high potency and extended duration of action), concerns over the possible toxicol. consequences of protein haptenization have hindered the development of the TCI concept. Recently, approaches to mitigate the risk of serious adverse reactions to this new class of agent have emerged, thus stimulating interest in the field and leading to authorization of the first cadre of TCIs to be marketed. The covalent inhibitor approach is rapidly gaining acceptance as a valuable tool in drug discovery, and is poised to make a major impact on the design of enzyme inhibitors and receptor modulators.
- 32Hallenbeck, K. K.; Turner, D. M.; Renslo, A. R.; Arkin, M. R. Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery. Curr. Top. Med. Chem. 2016, 17, 4– 15, DOI: 10.2174/1568026616666160719163839
- 33Lagoutte, R.; Patouret, R.; Winssinger, N. Covalent Inhibitors: An Opportunity for Rational Target Selectivity. Curr. Opin. Chem. Biol. 2017, 39, 54– 63, DOI: 10.1016/j.cbpa.2017.05.008[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpslGmsb4%253D&md5=2f7fac86ca6f6963050e1d6a69ef453eCovalent inhibitors: an opportunity for rational target selectivityLagoutte, Roman; Patouret, Remi; Winssinger, NicolasCurrent Opinion in Chemical Biology (2017), 39 (), 54-63CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)There is a resurging interest in compds. that engage their target through covalent interactions. Cysteine's thiol is endowed with enhanced reactivity, making it the nucleophile of choice for covalent engagement with a ligand aligning an electrophilic trap with a cysteine residue in a target of interest. The paucity of cysteine in the proteome coupled to the fact that closely related proteins do not necessarily share a given cysteine residue enable a level of unprecedented rational target selectivity. The recent demonstration that a lysine's amine can also be engaged covalently with a mild electrophile extends the potential of covalent inhibitors. The growing database of protein structures facilitates the discovery of covalent inhibitors while the advent of proteomic technologies enables a finer resoln. in the selectivity of covalently engaged proteins. Here, we discuss recent examples of discovery and design of covalent inhibitors.
- 34De Cesco, S.; Kurian, J.; Dufresne, C.; Mittermaier, A. K.; Moitessier, N. Covalent Inhibitors Design and Discovery. Eur. J. Med. Chem. 2017, 138, 96– 114, DOI: 10.1016/j.ejmech.2017.06.019[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKhsLrI&md5=551c9a9c4bd69d4ad4d9156c315d5a94Covalent inhibitors design and discoveryDe Cesco, Stephane; Kurian, Jerry; Dufresne, Caroline; Mittermaier, Anthony K.; Moitessier, NicolasEuropean Journal of Medicinal Chemistry (2017), 138 (), 96-114CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)In the history of therapeutics, covalent drugs occupy a very distinct category. While representing a significant fraction of the drugs on the market, very few have been deliberately designed to interact covalently with their biol. target. In this review, the prevalence of covalent drugs will first be briefly covered, followed by an introduction to their mechanisms of action and more detailed discussions of their discovery and the development of safe and efficient covalent enzyme inhibitors. All stages of a drug discovery program will be covered, from target considerations to lead optimization, strategies to tune reactivity and computational methods. The goal of this article is to provide an overview of the field and to outline good practices that are needed for the proper assessment and development of covalent inhibitors as well as a good understanding of the potential and limitations of current computational methods for the design of covalent drugs.
- 35Chaikuad, A.; Koch, P.; Laufer, S. A.; Knapp, S. The Cysteinome of Protein Kinases as a Target in Drug Development. Angew. Chem., Int. Ed. 2018, 57 (16), 4372– 4385, DOI: 10.1002/anie.201707875[Crossref], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFGgs7c%253D&md5=311ba5ab53dc4a5dc1dc92ddd4b6d4c2The Cysteinome of Protein Kinases as a Target in Drug DevelopmentChaikuad, Apirat; Koch, Pierre; Laufer, Stefan A.; Knapp, StefanAngewandte Chemie, International Edition (2018), 57 (16), 4372-4385CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Drugs that function through covalent bond formation represent a considerable fraction of the authors' repository of effective medicines but safety concerns and the complexity of developing covalent inhibitors has rendered covalent targeting a less attractive strategy for rational drug design. The recent approval of four covalent kinase inhibitors and the development of highly potent covalent kinase probes with exceptional selectivity has raised significant interest in industry and academic research and validated the concept of covalent kinase targeting for clin. applications. The abundance of cysteines at diverse positions in and around the kinase active site suggests that a large fraction of kinases can be targeted by covalent inhibitors. Herein, the authors review recent developments of this rapidly growing area in kinase drug development and highlight the unique opportunities and challenges of this strategy.
- 36Zhao, Z.; Bourne, P. E. Progress with Covalent Small-Molecule Kinase Inhibitors. Drug Discovery Today 2018, 23 (3), 727– 735, DOI: 10.1016/j.drudis.2018.01.035[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslShurg%253D&md5=f97a19f53a72044812a778ee9351fe94Progress with covalent small-molecule kinase inhibitorsZhao, Zheng; Bourne, Philip E.Drug Discovery Today (2018), 23 (3), 727-735CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)With reduced risk of toxicity and high selectivity, covalent small-mol. kinase inhibitors (CSKIs) have emerged rapidly. Through the lens of structural system pharmacol., here we review this rapid progress by considering design strategies and the challenges and opportunities offered by current CSKIs.
- 37Lonsdale, R.; Ward, R. A. Structure-Based Design of Targeted Covalent Inhibitors. Chem. Soc. Rev. 2018, 47 (11), 3816– 3830, DOI: 10.1039/C7CS00220C[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVGiurs%253D&md5=a7e31b23cf8ecfcad1397ad10ea10b7bStructure-based design of targeted covalent inhibitorsLonsdale, Richard; Ward, Richard A.Chemical Society Reviews (2018), 47 (11), 3816-3830CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Covalent inhibition is a rapidly growing discipline within drug discovery. Many historical covalent inhibitors were discovered by serendipity, with such a mechanism of action often regarded as undesirable due to potential toxicity issues. Recent progress has seen a major shift in this outlook, as covalent inhibition shows promise for targets where previous efforts to identify non-covalent small mol. inhibitors have failed. Targeted covalent inhibitors (TCIs) can offer drug discovery scientists the ability to increase the potency and/or selectivity of small mol. inhibitors, by attachment of reactive functional groups designed to covalently bind to specific sites in a target. In this tutorial review we introduce the broader concept of covalent inhibition, discuss the potential benefits and challenges of such an approach, and provide an overview of the current status of the field. We also describe some strategies and computational tools to enable successful covalent drug discovery.
- 38Ferguson, F. M.; Gray, N. S. Kinase Inhibitors: The Road Ahead. Nat. Rev. Drug Discovery 2018, 17 (5), 353– 377, DOI: 10.1038/nrd.2018.21[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXks12ktbg%253D&md5=34e42911d8a70301905460c3507e737aKinase inhibitors: the road aheadFerguson, Fleur M.; Gray, Nathanael S.Nature Reviews Drug Discovery (2018), 17 (5), 353-377CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review. Receptor tyrosine kinase signaling pathways have been successfully targeted to inhibit proliferation and angiogenesis for cancer therapy. However, kinase deregulation has been firmly demonstrated to play an essential role in virtually all major disease areas. Kinase inhibitor drug discovery programs have recently broadened their focus to include an expanded range of kinase targets and therapeutic areas. In this Review, we provide an overview of the novel targets, biol. processes and disease areas that kinase-targeting small mols. are being developed against, highlight the assocd. challenges and assess the strategies and technologies that are enabling efficient generation of highly optimized kinase inhibitors.
- 39Pettinger, J.; Jones, K.; Cheeseman, M. D. Lysine-Targeting Covalent Inhibitors. Angew. Chem., Int. Ed. 2017, 56 (48), 15200– 15209, DOI: 10.1002/anie.201707630[Crossref], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslejtr3P&md5=4a6bb561e53c165fa3c7c85953bb4736Lysine-Targeting Covalent InhibitorsPettinger, Jonathan; Jones, Keith; Cheeseman, Matthew D.Angewandte Chemie, International Edition (2017), 56 (48), 15200-15209CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Targeted covalent inhibitors have gained widespread attention in drug discovery as a validated method to circumvent acquired resistance in oncol. This strategy exploits small-mol./protein crystal structures to design tightly binding ligands with appropriately positioned electrophilic warheads. While most focus has been on targeting binding-site cysteine residues, targeting nucleophilic lysine residues can also represent a viable approach to irreversible inhibition. However, owing to the basicity of the ε-amino group in lysine, this strategy generates a no. of specific challenges. Herein, the authors review the key principles for inhibitor design, give historical examples, and present recent developments that demonstrate the potential of lysine targeting for future drug discovery.
- 40Jackson, P. A.; Widen, J. C.; Harki, D. A.; Brummond, K. M. Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions. J. Med. Chem. 2017, 60 (3), 839– 885, DOI: 10.1021/acs.jmedchem.6b00788[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitV2rtbnM&md5=36d3be5bf688c4e4a8cf4bcf42073009Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition ReactionsJackson, Paul A.; Widen, John C.; Harki, Daniel A.; Brummond, Kay M.Journal of Medicinal Chemistry (2017), 60 (3), 839-885CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Although Michael acceptors display a potent and broad spectrum of bioactivity, they have largely been ignored in drug discovery because of their presumed indiscriminate reactivity. As such, a dearth of information exists relevant to the thiol reactivity of natural products and their analogs possessing this moiety. In the midst of recently approved acrylamide-contg. drugs, it is clear that a good understanding of the hetero-Michael addn. reaction and the relative reactivities of biol. thiols with Michael acceptors under physiol. conditions is needed for the design and use of these compds. as biol. tools and potential therapeutics. This Perspective provides information that will contribute to this understanding, such as kinetics of thiol addn. reactions, bioactivities, as well as steric and electronic factors that influence the electrophilicity and reversibility of Michael acceptors. This Perspective is focused on α,β-unsatd. carbonyls given their preponderance in bioactive natural products.
- 41Baslé, E.; Joubert, N.; Pucheault, M. Protein Chemical Modification on Endogenous Amino Acids. Chem. Biol. 2010, 17 (3), 213– 227, DOI: 10.1016/j.chembiol.2010.02.008[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjvFKhurY%253D&md5=82a023a2f4e96136205bdb19901bd401Protein Chemical Modification on Endogenous Amino AcidsBasle, Emmanuel; Joubert, Nicolas; Pucheault, MathieuChemistry & Biology (Cambridge, MA, United States) (2010), 17 (3), 213-227CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)A review. Chem. modification of protein is an arduous but fruitful task. Many chem. methods have been developed for such purpose by carefully balancing reactivity and selectivity. Now both chemists and biologists have in hand an arsenal of tools from which they can select a relevant reaction to tackle their problems. This review focuses on the various chem. transformations available for selective modification of proteins. It also provides a brief overview of some of their main applications, including detection of protein interactions, prepn. of bioconjugates, and protein microarrays.
- 42Boutureira, O.; Bernardes, G. J. L. Advances in Chemical Protein Modification. Chem. Rev. 2015, 115 (5), 2174– 2195, DOI: 10.1021/cr500399p[ACS Full Text], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtV2nu7k%253D&md5=d21a5407a59a7e12346c16e5db75ab91Advances in Chemical Protein ModificationBoutureira, Omar; Bernardes, Goncalo J. L.Chemical Reviews (Washington, DC, United States) (2015), 115 (5), 2174-2195CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Transition metal-free and -mediated approaches are covered.
- 43Shannon, D. A.; Weerapana, E. Covalent Protein Modification: The Current Landscape of Residue-Specific Electrophiles. Curr. Opin. Chem. Biol. 2015, 24, 18– 26, DOI: 10.1016/j.cbpa.2014.10.021[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFSlsrzF&md5=ef6f0fe9ed08ee0653fe0f19f87bdafcCovalent protein modification: the current landscape of residue-specific electrophilesShannon, D. Alexander; Weerapana, EranthieCurrent Opinion in Chemical Biology (2015), 24 (), 18-26CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Functional amino acids that play crit. roles in catalysis and regulation are known to display elevated nucleophilicity and can be selectively targeted for covalent modification by reactive electrophiles. Chem.-proteomic platforms, such as activity-based protein profiling (ABPP), exploit this reactivity by utilizing chem. probes to covalently modify active-site residues to inform on the functional state of enzymes within complex proteomes. These and other applications rely on the availability of a diverse array of electrophiles and detailed knowledge of the reactivity and amino-acid selectivity of these groups. Here, we survey the current landscape of electrophiles that covalently target various nucleophilic amino acids in proteins and highlight proteomic applications that have benefited from the unique properties of these electrophiles.
- 44Gunnoo, S. B.; Madder, A. Chemical Protein Modification through Cysteine. ChemBioChem 2016, 17 (7), 529– 553, DOI: 10.1002/cbic.201500667[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjvVyrsrY%253D&md5=275b9e4e5770811079411cb048f47427Chemical Protein Modification through CysteineGunnoo, Smita B.; Madder, AnnemiekeChemBioChem (2016), 17 (7), 529-553CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chem. modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance-both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chem. protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.
- 45Dondoni, A.; Marra, A. SuFEx: A Metal-Free Click Ligation for Multivalent Biomolecules. Org. Biomol. Chem. 2017, 15 (7), 1549– 1553, DOI: 10.1039/C6OB02458K[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFGgurY%253D&md5=fcad56172f56f0d9bc17146097479495SuFEx: a metal-free click ligation for multivalent biomoleculesDondoni, Alessandro; Marra, AlbertoOrganic & Biomolecular Chemistry (2017), 15 (7), 1549-1553CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)A review. The Sulfur(VI) Fluoride Exchange (SuFEx), revived by Sharpless and co-workers from an unrecognized state, is an emerging new click reaction that is based on the high reactivity of sulfonyl fluorides and fluorosulfates with suitable nucleophiles such as silyl ethers and amines. Hence, we comment in this Perspective on the use of SuFEx for the synthesis of a new family of sugar contg. sulfonamides from the reaction of a glycosylsulfonyl fluoride with aliph. amines. We also highlight the applications of SuFEx to multivalent scaffolds such as tetraamino- and tetrafluorosulfonyl-calixarene leading to sulfonamide-linked sugar and iminosugar clusters. Finally, we report on the chemoselective functionalization of bovine serum albumin (BSA) which, owing to the amino group of lysine moieties, reacted with SO2F2 to give a multivalent BSA-SO2F system. The PEGylation of the same protein by coupling with a PEG-fluorosulfate is described as well.
- 46deGruyter, J. N.; Malins, L. R.; Baran, P. S. Residue-Specific Peptide Modification: A Chemist’s Guide. Biochemistry 2017, 56 (30), 3863– 3873, DOI: 10.1021/acs.biochem.7b00536[ACS Full Text], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKisLfK&md5=cb0a794b5077461762fd9fa43f4f39c2Residue-Specific Peptide Modification: A Chemist's GuidedeGruyter, Justine N.; Malins, Lara R.; Baran, Phil S.Biochemistry (2017), 56 (30), 3863-3873CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. Advances in bioconjugation and native protein modification are appearing at a blistering pace, making it increasingly time consuming for practitioners to identify the best chem. method to modify a specific amino acid residue in a complex setting. The purpose of this perspective is to provide an informative, graphically rich manual highlighting significant advances in the field over the past decade. This guide will help triage candidate methods for peptide alteration, and will serve as a starting point for those seeking to solve longstanding challenges.
- 47Hoch, D. G.; Abegg, D.; Adibekian, A. Cysteine-Reactive Probes and Their Use in Chemical Proteomics. Chem. Commun. 2018, 54 (36), 4501– 4512, DOI: 10.1039/C8CC01485J[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnsVegs7k%253D&md5=d91bb3d96048cb6c9516d69033b2365fCysteine-reactive probes and their use in chemical proteomicsHoch, Dominic G.; Abegg, Daniel; Adibekian, AlexanderChemical Communications (Cambridge, United Kingdom) (2018), 54 (36), 4501-4512CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. Proteomic profiling using bioorthogonal chem. probes that selectively react with certain amino acids is now a widely used method in life sciences to investigate enzymic activities, study posttranslational modifications and discover novel covalent inhibitors. Over the past two decades, researchers have developed selective probes for several different amino acids, including lysine, serine, cysteine, threonine, tyrosine, aspartate and glutamate. Among these amino acids, cysteines are particularly interesting due to their highly diverse and complex biochem. role in our cells. In this feature article, we focus on the chem. probes and methods used to study cysteines in complex proteomes.
- 48Cromm, P. M.; Crews, C. M. The Proteasome in Modern Drug Discovery: Second Life of a Highly Valuable Drug Target. ACS Cent. Sci. 2017, 3 (8), 830– 838, DOI: 10.1021/acscentsci.7b00252[ACS Full Text], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1Orsb3M&md5=91f26fd1133db650c0b4b4f43a461354The Proteasome in Modern Drug Discovery: Second Life of a Highly Valuable Drug TargetCromm, Philipp M.; Crews, Craig M.ACS Central Science (2017), 3 (8), 830-838CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)A review. As the central figure of the cellular protein degrdn. machinery, the proteasome is crit. for cell survival. Having been extensively targeted for inhibition, the constitutive proteasome has proven its role as a highly valuable drug target. However, recent advances in the protein homeostasis field suggest that addnl. chapters can be added to this successful story. For example, selective immunoproteasome inhibition promises high clin. efficacy for autoimmune disorders and inflammation, and proteasome inhibitors might serve as novel therapeutics for malaria or other microorganisms. Furthermore, utilizing the destructive force of the proteasome for selective degrdn. of essential drivers of human disorders has opened up a new and exciting area of drug discovery. Thus, the field of proteasome drug discovery still holds exciting questions to be answered and does not simply end with inhibiting the constitutive proteasome.
- 49Casimiro-Garcia, A.; Trujillo, J. I.; Vajdos, F.; Juba, B.; Banker, M. E.; Aulabaugh, A.; Balbo, P.; Bauman, J.; Chrencik, J.; Coe, J. W.; Czerwinski, R.; Dowty, M.; Knafels, J. D.; Kwon, S.; Leung, L.; Liang, S.; Robinson, R. P.; Telliez, J.-B.; Unwalla, R.; Yang, X.; Thorarensen, A. Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors. J. Med. Chem. 2018, 61, 10665– 10699, DOI: 10.1021/acs.jmedchem.8b01308[ACS Full Text], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitF2qtrjK&md5=12832ed66b8fc89d5f6ef9a3aa4ea20fIdentification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent InhibitorsCasimiro-Garcia, Agustin; Trujillo, John I.; Vajdos, Felix; Juba, Brian; Banker, Mary Ellen; Aulabaugh, Ann; Balbo, Paul; Bauman, Jonathan; Chrencik, Jill; Coe, Jotham W.; Czerwinski, Robert; Dowty, Martin; Knafels, John D.; Kwon, Soojin; Leung, Louis; Liang, Sidney; Robinson, Ralph P.; Telliez, Jean-Baptiste; Unwalla, Ray; Yang, Xin; Thorarensen, AtliJournal of Medicinal Chemistry (2018), 61 (23), 10665-10699CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Ongoing interest in the discovery of selective JAK3 inhibitors led us to design novel covalent inhibitors that engage the JAK3 residue Cys909 by cyanamide, a structurally and mechanistically differentiated electrophile from other cysteine reacting groups previously incorporated in JAK3 covalent inhibitors. Through crystallog., kinetic, and computational studies, interaction of cyanamide I with Cys909 was optimized leading to potent and selective JAK3 inhibitors as exemplified by II. In relevant cell-based assays and in agreement with previous results from this group, II demonstrated that selective inhibition of JAK3 is sufficient to drive JAK1/JAK3-mediated cellular responses. The contribution from extrahepatic processes to the clearance of cyanamide-based covalent inhibitors was also characterized using metabolic and pharmacokinetic data for I. This work also gave key insights into a productive approach to decrease glutathione/glutathione S-transferase-mediated clearance, a challenge typically encountered during the discovery of covalent kinase inhibitors.
- 50Li, T.; Maltais, R.; Poirier, D.; Lin, S.-X. Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide. J. Phys. Chem. Lett. 2018, 9 (18), 5275– 5280, DOI: 10.1021/acs.jpclett.8b02225[ACS Full Text], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1SitbbJ&md5=46fc3f11f2484b972c5b6ce626136e14Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl HalideLi, Tang; Maltais, Rene; Poirier, Donald; Lin, Sheng-XiangJournal of Physical Chemistry Letters (2018), 9 (18), 5275-5280CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) plays a pivotal role in the progression of estrogen-related diseases because of its involvement in the biosynthesis of estradiol (E2), constituting a valuable therapeutic target for endocrine treatment. In the present study, we successfully cocrystd. the enzyme with the reversible inhibitor 2-methoxy-16β-(m-carbamoylbenzyl)-E2 (2-MeO-CC-156) as well as the enzyme with the irreversible inhibitor 3-(2-bromoethyl)-16β-(m-carbamoylbenzyl)-17β-hydroxy-1,3,5(10)-estratriene (PBRM). The structures of ternary complexes of 17β-HSD1-2-MeO-CC-156-NADP+ and 17β-HSD1-PBRM-NADP+ comparatively show the formation of a covalent bond between His221 and the bromoethyl side chain of the inhibitor in the PBRM structure. A dynamic process including beneficial mol. interactions that favor the specific binding of a low-reactivity inhibitor and subsequent N-alkylation event through the participation of His221 in the enzyme catalytic site clearly demonstrates the covalent bond formation. This finding opens the door to a new design of alkyl halide-based specific covalent inhibitors as potential therapeutic agents for different enzymes, contributing to the development of highly efficient inhibitors.
- 51Kharenko, O. A.; Patel, R. G.; Brown, S. D.; Calosing, C.; White, A.; Lakshminarasimhan, D.; Suto, R. K.; Duffy, B. C.; Kitchen, D. B.; McLure, K. G.; Hansen, H. C.; van der Horst, E. H.; Young, P. R. Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine. J. Med. Chem. 2018, 61 (18), 8202– 8211, DOI: 10.1021/acs.jmedchem.8b00666[ACS Full Text], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1ensLjO&md5=1c3ae393a5f8a8c12ffb16e6f3798118Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a MethionineKharenko, Olesya A.; Patel, Reena G.; Brown, S. David; Calosing, Cyrus; White, Andre; Lakshminarasimhan, Damodharan; Suto, Robert K.; Duffy, Bryan C.; Kitchen, Douglas B.; McLure, Kevin G.; Hansen, Henrik C.; van der Horst, Edward H.; Young, Peter R.Journal of Medicinal Chemistry (2018), 61 (18), 8202-8211CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small mol. bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacol. Here the authors describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an x-ray crystal structure, the authors demonstrate that these inhibitors selectively form a covalent bond with Met 149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.
- 52Pearson, R. G.; Sobel, H. R.; Songstad, J. Nucleophilic Reactivity Constants toward Methyl Iodide and Trans-Dichlorodi(Pyridine)Platinum(II). J. Am. Chem. Soc. 1968, 90 (2), 319– 326, DOI: 10.1021/ja01004a021[ACS Full Text], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF1cXhtFOntbk%253D&md5=b118d94887322fd7cdda64874f873aadNucleophilic reactivity constants toward methyl iodide and trans-dichlorodi(pyridine)platinum(II)Pearson, Ralph G.; Sobel, Harold R.; Songstad, JonJournal of the American Chemical Society (1968), 90 (2), 319-26CODEN: JACSAT; ISSN:0002-7863.The rates of reaction of a no. of nucleophiles with MeI and trans-[Pt(py)2Cl2] have been measured in MeOH at 25°. Relative nucleophilic reactivity parameters, nMeI and nPt, have been calcd. It was not possible to correlate these nos. with each other or with other extra-kinetic data. Equations in the literature for predicting nucleophilic reactivity have only a limited range of usefulness. 32 references.
- 53Sardi, F.; Manta, B.; Portillo-Ledesma, S.; Knoops, B.; Comini, M. A.; Ferrer-Sueta, G. Determination of Acidity and Nucleophilicity in Thiols by Reaction with Monobromobimane and Fluorescence Detection. Anal. Biochem. 2013, 435 (1), 74– 82, DOI: 10.1016/j.ab.2012.12.017[Crossref], [PubMed], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXisVGlsbk%253D&md5=72eb72b04f8626d6cd592ddeb60f779dDetermination of acidity and nucleophilicity in thiols by reaction with monobromobimane and fluorescence detectionSardi, Florencia; Manta, Bruno; Portillo-Ledesma, Stephanie; Knoops, Bernard; Comini, Marcelo A.; Ferrer-Sueta, GerardoAnalytical Biochemistry (2013), 435 (1), 74-82CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)A method based on the differential reactivity of thiol and thiolate with monobromobimane (mBBr) has been developed to measure nucleophilicity and acidity of protein and low-mol.-wt. thiols. Nucleophilicity of the thiolate is measured as the pH-independent second-order rate const. of its reaction with mBBr. The ionization consts. of the thiols are obtained through the pH dependence of either second-order rate const. or initial rate of reaction. For readily available thiols, the apparent second-order rate const. is measured at different pHs and then plotted and fitted to an appropriate pH function describing the obsd. no. of ionization equil. For less available thiols, such as protein thiols, the initial rate of reaction is detd. in a wide range of pHs and fitted to the appropriate pH function. The method presented here shows excellent sensitivity, allowing the use of nanomolar concns. of reagents. The method is suitable for scaling and high-throughput screening. Example detns. of nucleophilicity and pKa are presented for captopril and cysteine as low-mol.-wt. thiols and for human peroxiredoxin 5 and Trypanosoma brucei monothiol glutaredoxin 1 as protein thiols.
- 54Pearson, R. G. Hard and Soft Acids and Bases. J. Am. Chem. Soc. 1963, 85 (22), 3533– 3539, DOI: 10.1021/ja00905a001[ACS Full Text], [CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2cXksV0%253D&md5=2333a18c8168b373b612f5f45e8f4b4cHard and soft acids and basesPearson, Ralph G.Journal of the American Chemical Society (1963), 85 (22), 3533-9CODEN: JACSAT; ISSN:0002-7863.A number of Lewis acids of diverse types are classified as (a) or (b) following the criterion of Ahrland, et al. (CA 53, 960c). Other, auxiliary criteria are proposed. Class (a) acids prefer to bind to "hard" or nonpolarizable bases. Class (b) acids prefer to bind to "soft" or polarizable bases. Since class (a) acids are themselves "hard" and since class (b) acids are "soft" a simple, useful rule is proposed: hard acids bind strongly to hard bases and soft acids bind strongly to soft bases. The explanations for such behavior include: (1) various degrees of ionic and covalent σ-bonding; (2) π-bonding; (3) electron correlation phenomena; (4) solvation effects.
- 55Awoonor-Williams, E.; Rowley, C. N. Evaluation of Methods for the Calculation of the PKa of Cysteine Residues in Proteins. J. Chem. Theory Comput. 2016, 12 (9), 4662– 4673, DOI: 10.1021/acs.jctc.6b00631[ACS Full Text], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlOqtbvM&md5=f2d43fd808bdfb63fe2b19639ab4e64cEvaluation of Methods for the Calculation of the pKa of Cysteine Residues in ProteinsAwoonor-Williams, Ernest; Rowley, Christopher N.Journal of Chemical Theory and Computation (2016), 12 (9), 4662-4673CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)Methods for the calcn. of the pK of ionizable amino acids are valuable tools for understanding pH-dependent properties of proteins. Cysteine is unique among the amino acids because of the chem. reactivity of its thiol group (S-H), which plays an instrumental role in several biochem. and regulatory functions. The acidity of noncatalytic cysteine residues is a factor in their susceptibility to chem. modification. Despite the plethora of existing pKa computing methods, no definitive protocol exists for accurately calcg. the pKa's of cysteine residues in proteins. A cysteine pKa test set was developed, which is comprised of 18 cysteine residues in 12 proteins where the pKa's have been detd. exptl. and an exptl. structure is available. The pKa's of these residues were calcd. using three methods that use an implicit solvent model (H++, MCCE, and PROPKA) and an all-atom replica exchange thermodn. integration approach with the CHARMM36 and AMBER ff99SB-ILDNP force fields. The models that use implicit solvation methods were generally unreliable in predicting cysteine residue pKa's, with RMSDs between 3.41 and 4.72 pKa units. On av., the explicit solvent methods performed better than the implicit solvent methods. RMSD values of 2.40 and 3.20 were obtained for simulations with the CHARMM36 and AMBER ff99SB-ILDNP force fields, resp. Further development of these methods is necessary because the performance of the best method is similar to that of the null-model (RMSD = 2.74) and these differences in RMSD are of limited statistical significance given the small size of the authors' test set.
- 56Grimsley, G. R.; Scholtz, J. M.; Pace, C. N. A Summary of the Measured PK Values of the Ionizable Groups in Folded Proteins. Protein Sci. 2008, 18, 247– 251, DOI: 10.1002/pro.19
- 57Alcock, L. J.; Perkins, M. V.; Chalker, J. M. Chemical Methods for Mapping Cysteine Oxidation. Chem. Soc. Rev. 2018, 47 (1), 231– 268, DOI: 10.1039/C7CS00607A[Crossref], [PubMed], [CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFyisbvE&md5=498906bd9e79704dfd5ffd78791d25caChemical methods for mapping cysteine oxidationAlcock, Lisa J.; Perkins, Michael V.; Chalker, Justin M.Chemical Society Reviews (2018), 47 (1), 231-268CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Cysteine residues in proteins are subject to diverse redox chem. Oxidn. of cysteine to S-nitrosocysteine, cysteine sulfenic and sulfinic acids, disulfides and persulfides are a few prominent examples of these oxidative post-translational modifications. In living organisms, these modifications often play key roles in cell signalling and protein function, but a full account of this biochem. is far from complete. It is therefore an important goal in chem. biol. to identify what proteins are subjected to these modifications and understand their physiol. function. This review provides an overview of these modifications, how they can be detected and quantified using chem. probes, and how this information provides insight into their role in biol. This survey also highlights future opportunities in the study of cysteine redox chem., the challenges that await chemists and biologists in this area of study, and how meeting such challenges might reveal valuable information for biomedical science.
- 58Pace, N. J.; Weerapana, E. Diverse Functional Roles of Reactive Cysteines. ACS Chem. Biol. 2013, 8 (2), 283– 296, DOI: 10.1021/cb3005269[ACS Full Text], [CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1yksrfE&md5=2d27df87f153dd9c916dc901f9570644Diverse Functional Roles of Reactive CysteinesPace, Nicholas J.; Weerapana, EranthieACS Chemical Biology (2013), 8 (2), 283-296CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A review. Cysteine residues on proteins play key roles in catalysis and regulation. These functional cysteines serve as active sites for nucleophilic and redox catalysis, sites of allosteric regulation, and metal-binding ligands on proteins from diverse classes including proteases, kinases, metabolic enzymes, and transcription factors. In this review, we focus on a few select examples that serve to highlight the multiple functions performed by cysteines, with an emphasis on cysteine-mediated protein activities implicated in cancer. The enhanced reactivity of functional cysteines renders them susceptible to modification by electrophilic species. Toward this end, we discuss recent advancements and future prospects for utilizing cysteine-reactive small mols. as drugs and imaging agents for the treatment and diagnosis of cancer.
- 59Jones, A.; Zhang, X.; Lei, X. Covalent Probe Finds Carboxylic Acid. Cell Chem. Biol. 2017, 24 (5), 537– 539, DOI: 10.1016/j.chembiol.2017.05.003[Crossref], [PubMed], [CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXotlOqtLg%253D&md5=8f3451424653935cc1d168faec24c9a6Covalent Probe Finds Carboxylic AcidJones, Alexander; Zhang, Xiaoyun; Lei, XiaoguangCell Chemical Biology (2017), 24 (5), 537-539CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)In this issue of Cell Chem. Biol., Marti´n-Gago et al. (2017a) disclose a new strategy for the selective covalent targeting of binding site carboxylic acids within the proteome using the isoxazolium salt as a warhead. This discovery paves the way for developing new protein ligation methods as well as covalent drug candidates.
- 60Shirley, M. Dacomitinib: First Global Approval. Drugs 2018, 78, 1947, DOI: 10.1007/s40265-018-1028-x[Crossref], [PubMed], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3crnslGgsw%253D%253D&md5=0e035cd4b47b60f40c2af1d3c1fb5addDacomitinib: First Global ApprovalShirley MattDrugs (2018), 78 (18), 1947-1953 ISSN:.Dacomitinib (Vizimpro(®)) is an orally administered, small-molecule irreversible inhibitor of HER1 (EGFR), HER2 and HER4 that was developed by Pfizer Inc. for the treatment of solid tumours. In September 2018, dacomitinib received its first global approval, in the USA, for use in the first-line treatment of patients with metastatic NSCLC with EGFR exon 19 deletion or exon 21 L858R substitution mutations as detected by an FDA-approved test. Registration applications for the use of dacomitinib as first-line treatment for patients with EGFR-mutation-positive metastatic NSCLC have also been submitted in the EU and Japan. This article summarizes the milestones in the development of dacomitinib leading to this first approval for the first-line treatment of patients with EGFR-mutated metastatic NSCLC.
- 61Markham, A.; Dhillon, S. Acalabrutinib: First Global Approval. Drugs 2018, 78 (1), 139– 145, DOI: 10.1007/s40265-017-0852-8[Crossref], [PubMed], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFekurvF&md5=95ac3e46917e6e1b248a1a1da537f562Acalabrutinib: First Global ApprovalMarkham, Anthony; Dhillon, SohitaDrugs (2018), 78 (1), 139-145CODEN: DRUGAY; ISSN:0012-6667. (Springer International Publishing AG)Acerta Pharma is developing the Bruton's tyrosine kinase inhibitor acalabrutinib (Calquence) for the treatment of various haematol. and solid malignancies. The drug has received accelerated approval from the US FDA for the treatment of mantle cell lymphoma based on the results of a phase II study, and phase III trials in mantle cell lymphoma and chronic lymphocytic leukemia are currently underway. This article summarizes the milestones in the development of acalabrutinib leading to this first approval for mantle cell lymphoma.
- 62Forster, M.; Gehringer, M.; Laufer, S. A. Recent Advances in JAK3 Inhibition: Isoform Selectivity by Covalent Cysteine Targeting. Bioorg. Med. Chem. Lett. 2017, 27 (18), 4229– 4237, DOI: 10.1016/j.bmcl.2017.07.079[Crossref], [PubMed], [CAS], Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtl2lsb3M&md5=3c6b2f86ccd3e3f3e254bbece5fa3ac5Recent advances in JAK3 inhibition: Isoform selectivity by covalent cysteine targetingForster, Michael; Gehringer, Matthias; Laufer, Stefan A.Bioorganic & Medicinal Chemistry Letters (2017), 27 (18), 4229-4237CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Janus kinases (JAKs) are a family of four cytosolic protein kinases with a high degree of structural similarity. Due to its very restricted role in immune regulation, JAK3 was promoted as an excellent target for immunosuppression for more than a decade, but clin. validation of this concept is still elusive. During the last years, speculation arose that kinase activity of JAK1, which cooperates with JAK3 in cytokine receptor signaling, may have a dominant role over the one of JAK3. Until recently, however, this issue could not be appropriately addressed due to a lack of highly isoform-selective tool compds. With the recent resurgence of covalent drugs, targeting of a specific cysteine that distinguishes JAK3 from other JAK family members became an attractive design option. By applying this strategy, a set of JAK3 inhibitors with excellent selectivity against other JAK isoforms and the kinome was developed during the last three years and used to decipher JAK3-dependent signaling. The data obtained with these tool compds. demonstrates that selective JAK3 inhibition is sufficient to block downstream signaling. Since one of these inhibitors is currently under evaluation in phase II clin. studies against several inflammatory disorders, it will soon become apparent whether selective JAK3 inhibition translates into clin. efficacy.
- 63Garzón, B.; Oeste, C. L.; Díez-Dacal, B.; Pérez-Sala, D. Proteomic Studies on Protein Modification by Cyclopentenone Prostaglandins: Expanding Our View on Electrophile Actions. J. Proteomics 2011, 74 (11), 2243– 2263, DOI: 10.1016/j.jprot.2011.03.028[Crossref], [PubMed], [CAS], Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlKit77P&md5=b7c68bfbcdd4bcebef40a03b82703d19Proteomic studies on protein modification by cyclopentenone prostaglandins: Expanding our view on electrophile actionsGarzon, Beatriz; Oeste, Clara L.; Diez-Dacal, Beatriz; Perez-Sala, DoloresJournal of Proteomics (2011), 74 (11), 2243-2263CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)A review. Cyclopentenone prostaglandins (cyPG) are lipid mediators that participate in the mechanisms regulating inflammation and tumorigenesis. The cyPG are electrophilic compds. that act mainly through the covalent modification of cellular proteins. The stability of many cyPG-protein adducts makes them suitable for proteomic anal. Indeed, methodol. advances in recent years have allowed identifying many cyPG targets, including components of pro-inflammatory transcription factors, cytoskeletal proteins, signaling kinases and proteins involved in redox control. Insight into the diversity of cyPG targets is providing a better understanding of their mechanism of action, uncovering novel links between resoln. of inflammation, proliferation and redox regulation. Moreover, identification of the target residues has unveiled the selectivity of protein modification by these electrophiles, providing valuable information for potential pharmacol. applications. Among the challenges ahead, the detection of proteins modified by endogenous cyPG and the quant. aspects of the modification require further efforts. Importantly, only a few years after the appearance of the first proteomic studies, research on cyPG targets is yielding new paradigms for redox and electrophilic signaling.
- 64Zhao, Z.; Liu, Q.; Bliven, S.; Xie, L.; Bourne, P. E. Determining Cysteines Available for Covalent Inhibition Across the Human Kinome. J. Med. Chem. 2017, 60 (7), 2879– 2889, DOI: 10.1021/acs.jmedchem.6b01815[ACS Full Text], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXkvVGjsLg%253D&md5=22ea95ce38eb0836d32c1332dba54fadDetermining Cysteines Available for Covalent Inhibition Across the Human KinomeZhao, Zheng; Liu, Qingsong; Bliven, Spencer; Xie, Lei; Bourne, Philip E.Journal of Medicinal Chemistry (2017), 60 (7), 2879-2889CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Covalently bound protein kinase inhibitors have been frequently designed to target non-catalytic Cys residues at the ATP binding site. Thus, it is important to know if a given Cys residue can form a covalent bond. Here, we combined a function-site interaction fingerprint method and DFT calcns. to det. the potential of Cys residues to form a covalent interaction with an inhibitor. By harnessing the human structural kinome, a comprehensive structure-based binding site Cys dataset was assembled. The orientation of the Cys SH group indicated which Cys residues could potentially form covalent bonds. These covalent inhibitor accessible Cys residues were located within 5 kinase regions (P-loop, roof of pocket, front pocket, catalytic-2 of the catalytic loop and DFG-3 close to the DFG peptide). In an independent test set, these Cys residues covered 95% of covalent kinase inhibitors. Thus, this study provides new insights into Cys reactivity and preference which is important for the prospective development of covalent kinase inhibitors.
- 65Günther, M.; Juchum, M.; Kelter, G.; Fiebig, H.; Laufer, S. Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S Mutant. Angew. Chem., Int. Ed. 2016, 55 (36), 10890– 10894, DOI: 10.1002/anie.201603736[Crossref], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1GmtbzK&md5=c3909faa7f781c2a3679f75bd3b6c5acLung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S MutantGuenther, Marcel; Juchum, Michael; Kelter, Gerhard; Fiebig, Heiner; Laufer, StefanAngewandte Chemie, International Edition (2016), 55 (36), 10890-10894CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The treatment of non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) inhibitors is made challenging by acquired resistance caused by somatic mutations. Third-generation EGFR inhibitors have been designed to overcome resistance through covalent binding to the Cys 797 residue of the enzyme, and these inhibitors are effective against most clin. relevant EGFR mutants. However, the high dependence of these recent EGFR inhibitors on this particular interaction means that addnl. mutation of Cys 797 results in poor inhibitory activity, which leads to tumor relapse in initially responding patients. A new generation of irreversible and reversible mutant EGFR inhibitors was developed with strong noncovalent binding properties, and these compds. show high inhibitory activities against the cysteine-mutated L858R/T790M/C797S EGFR.
- 66Niessen, S.; Dix, M. M.; Barbas, S.; Potter, Z. E.; Lu, S.; Brodsky, O.; Planken, S.; Behenna, D.; Almaden, C.; Gajiwala, K. S.; Ryan, K.; Ferre, R.; Lazear, M. R.; Hayward, M. M.; Kath, J. C.; Cravatt, B. F. Proteome-Wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors. Cell Chem. Biol. 2017, 24, 1388– 1400, DOI: 10.1016/j.chembiol.2017.08.017[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFOrtrnL&md5=dc2db6d29932ffdb991027ba272bd149proteome-wide map of targets of t790m-egfr-directed covalent inhibitorsNiessen, Sherry; Dix, Melissa M.; Barbas, Sabrina; Potter, Zachary E.; Lu, Shuyan; Brodsky, Oleg; Planken, Simon; Behenna, Douglas; Almaden, Chau; Gajiwala, Ketan S.; Ryan, Kevin; Ferre, RoseAnn; Lazear, Michael R.; Hayward, Matthew M.; Kath, John C.; Cravatt, Benjamin F.Cell Chemical Biology (2017), 24 (11), 1388-1400.e7CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chem. proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chem. proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety.
- 67Blewett, M. M.; Xie, J.; Zaro, B. W.; Backus, K. M.; Altman, A.; Teijaro, J. R.; Cravatt, B. F. Chemical Proteomic Map of Dimethyl Fumarate–Sensitive Cysteines in Primary Human T Cells. Sci. Signaling 2016, 9 (445), rs10, DOI: 10.1126/scisignal.aaf7694
- 68Deeks, E. D. Ibrutinib: A Review in Chronic Lymphocytic Leukaemia. Drugs 2017, 77 (2), 225– 236, DOI: 10.1007/s40265-017-0695-3[Crossref], [PubMed], [CAS], Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht12lurg%253D&md5=01ea759b722431401e1e1bb85e1f87c8Ibrutinib: A Review in Chronic Lymphocytic LeukaemiaDeeks, Emma D.Drugs (2017), 77 (2), 225-236CODEN: DRUGAY; ISSN:0012-6667. (Springer International Publishing AG)Ibrutinib (Imbruvica) is an oral irreversible inhibitor of Bruton's tyrosine kinase, a B-cell receptor (BCR) signalling kinase expressed by various hematopoietic cells, B-cell lymphomas and leukemias. The drug is indicated for the treatment of certain haematol. malignancies, including chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), which are the focus of this review. In phase III CLL/SLL trials, ibrutinib monotherapy was more effective than chlorambucil in the first-line treatment of elderly patients (RESONATE-2) and more effective than ofatumumab in previously-treated adults (RESONATE). Likewise, a combination of ibrutinib, bendamustine and rituximab was more effective in previously-treated adults than bendamustine plus rituximab in a phase III placebo-controlled study (HELIOS). These ibrutinib regimens were assocd. with significantly better progression-free survival, overall response rates, and overall survival than the comparators (in protocol-specified or planned analyses), with ibrutinib therapy providing benefit regardless of adverse prognostic factors, such as del(17p)/TP53 mutation and del(11q). Ibrutinib has an acceptable tolerability profile, although certain adverse events (e.g. bleeding and atrial fibrillation) require consideration. Redistribution lymphocytosis can occur, but is not indicative of disease progression. Although longer-term data would be beneficial, ibrutinib is a welcome treatment option for patients with CLL, including those who have higher-risk disease or are less phys. fit. Indeed, current EU and US guidelines recommend/prefer the drug for the first- and/or subsequent-line treatment of certain patients, including those with del(17p)/TP53 mutation.
- 69Bender, A. T.; Gardberg, A.; Pereira, A.; Johnson, T.; Wu, Y.; Grenningloh, R.; Head, J.; Morandi, F.; Haselmayer, P.; Liu-Bujalski, L. Ability of Bruton’s Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but Not B-Cell Receptor Signaling. Mol. Pharmacol. 2017, 91 (3), 208– 219, DOI: 10.1124/mol.116.107037[Crossref], [PubMed], [CAS], Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpsFajtr8%253D&md5=f869d52d6bc1aabf352988787ff1cbcfAbility of Bruton's tyrosine kinase inhibitors to sequester Y551 and prevent phosphorylation determines potency for inhibition of Fc receptor but not B-cell receptor signalingBender, Andrew T.; Gardberg, Anna; Pereira, Albertina; Johnson, Theresa; Wu, Yin; Grenningloh, Roland; Head, Jared; Morandi, Federica; Haselmayer, Philipp; Liu-Bujalski, LesleyMolecular Pharmacology (2017), 91 (3), 208-219CODEN: MOPMA3; ISSN:1521-0111. (American Society for Pharmacology and Experimental Therapeutics)Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large no. of Btk inhibitors have been developed. These compds. have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a mol. level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compds. can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcγR signaling as Y551 sequestering compds. were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in detg. potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compds. with a broader inhibitory profile that will likely result in greater efficacy.
- 70Pan, Z.; Scheerens, H.; Li, S.-J.; Schultz, B. E.; Sprengeler, P. A.; Burrill, L. C.; Mendonca, R. V.; Sweeney, M. D.; Scott, K. C. K.; Grothaus, P. G.; Jeffery, D. A.; Spoerke, J. M.; Honigberg, L. A.; Young, P. R.; Dalrymple, S. A.; Palmer, J. T. Discovery of Selective Irreversible Inhibitors for Bruton’s Tyrosine Kinase. ChemMedChem 2007, 2 (1), 58– 61, DOI: 10.1002/cmdc.200600221[Crossref], [PubMed], [CAS], Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjsVWjsbg%253D&md5=023ba8bca9942ad0127d7cf32293e9d2Discovery of selective irreversible inhibitors for bruton's tyrosine kinasePan, Zhengying; Scheerens, Heleen; Li, Shyr-Jiann; Schultz, Brian E.; Sprengeler, Paul A.; Burrill, L. Chuck; Mendonca, Rohan V.; Sweeney, Michael D.; Scott, Keana C. K.; Grothaus, Paul G.; Jeffery, Douglas A.; Spoerke, Jill M.; Honigberg, Lee A.; Young, Peter R.; Dalrymple, Stacie A.; Palmer, James T.ChemMedChem (2007), 2 (1), 58-61CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)Synthesis and pharmacol. evaluation of a series of peptidomimetic quinoline derivs. was undertaken to evaluate their efficacy in acting as selective irreversible inhibitors of Bruton's tyrosine kinase (Btk). With the relative scarcity of knowledge on the inhibition of Btk it is crucial to discover a potent and selective tool compd. for this kinase. Herein is described the discovery of selective irreversible Btk inhibitors and their efficacy in a mouse RA model.
- 71Mann, M. Innovations: Functional and Quantitative Proteomics Using SILAC. Nat. Rev. Mol. Cell Biol. 2006, 7 (12), 952– 958, DOI: 10.1038/nrm2067[Crossref], [PubMed], [CAS], Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1KisrrF&md5=4e658b29b1c6e91abf33f86babaf93c1Functional and quantitative proteomics using SILACMann, MatthiasNature Reviews Molecular Cell Biology (2006), 7 (12), 952-958CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Researchers in many biol. areas now routinely characterize proteins by mass spectrometry. Among the many formats for quant. proteomics, stable-isotope labeling by amino acids in cell culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quant. phosphoproteomics technol. - directly uncovers important points in the signalling pathways that control cellular decisions.
- 72Crow, J. A.; Bittles, V.; Borazjani, A.; Potter, P. M.; Ross, M. K. Covalent Inhibition of Recombinant Human Carboxylesterase 1 and 2 and Monoacylglycerol Lipase by the Carbamates JZL184 and URB597. Biochem. Pharmacol. 2012, 84 (9), 1215– 1222, DOI: 10.1016/j.bcp.2012.08.017[Crossref], [PubMed], [CAS], Google Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlertLjF&md5=02eb736350283cc8b9e6224cd9f3351dCovalent inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by the carbamates JZL184 and URB597Crow, J. Allen; Bittles, Victoria; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.Biochemical Pharmacology (2012), 84 (9), 1215-1222CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metab. of several pharmaceuticals. Recently, carbamate compds. were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resp.; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate consts. for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimol. rate consts. (kinact/Ki) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 103 M-1 s-1 and 4.5 (±1.3) × 103 M-1 s-1, resp.]. However, kinact/Ki for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 102 M-1 s-1 and 3.9 (±1.0) × 103 M-1 s-1, resp.]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also detd. kinetic consts. for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metab.
- 73Buynak, J. D.; Mathew, J.; Rao, M. N.; Haley, E.; George, C.; Siriwardane, U. The Preparation of the First α-Vinylidene-β-Lactams. J. Chem. Soc., Chem. Commun. 1987, 0 (10), 735– 737, DOI: 10.1039/C39870000735[Crossref], [CAS], Google Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXlsFOjug%253D%253D&md5=d65839405f355c0236d626cd95eeb012The preparation of the first α-vinylidene-β-lactamsBuynak, John D.; Mathew, Jacob; Rao, M. Narayana; Haley, Elizabeth; George, Christian; Siriwardane, UpaliJournal of the Chemical Society, Chemical Communications (1987), (10), 735-7CODEN: JCCCAT; ISSN:0022-4936.α-Vinylidene-β-lactams I (R = H, R1 = Me, CH2Ph; R = Me, CH2Ph, CH2C6H4Br-4, CH2C6H4OMe-4, H, R1 = Me) were prepd. from MeCH:C:CHSC6H4Cl-4 and chlorosulfonyl isocyanate. Crystal structure of I (R = H, R1 = CH2Ph) was reported. β-Lactam II was prepd. similarly.
- 74Roedig, A.; Ritschel, W. Reaktionen von 3,4,4-Trichlor-3-butenamiden mit Nucleophilen, II. Thiol- und Aminaddukte von 3,3-Dichlorallencarboxamiden. Chem. Ber. 1983, 116 (4), 1595– 1602, DOI: 10.1002/cber.19831160434[Crossref], [CAS], Google Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXktVGitLc%253D&md5=7ac804c6a6bfcb66fff16d78f4968336Reactions of 3,4,4-trichloro-3-butenamides with nucleophiles. II. Thiol- and amine adducts of 3,3-dichloroallenecarboxamidesRoedig, Alfred; Ritschel, WernerChemische Berichte (1983), 116 (4), 1595-602CODEN: CHBEAM; ISSN:0009-2940.Treatment of Cl2C:C:CHCONHR (I, R = 4-MeC6H4, Me3C, Me2CH), prepd. by dehydrochlorination of Cl2C:CClCH2CONHR, with R1R2NH (R1 = R2 = Me, Et; R1 = Me, R2 = Ph; R1 = Me3C, Me2CH, Pr, R2 = H; R1R2N = morpholino) gave the corresponding adducts Cl2CHC(NR1R2):CHCONHR. Treatment of I with R3S- (R3 = 4-ClC6H4, C6Cl5, Pr, Me3C) gave Cl2C:C(SR3)CH2CONHR (II) of which II (R = Me3C, R3 = Pr, Me3C) rearrange slowly in base to give Cl2CHC(SR3):CHCONHR.
- 75Abbas, A.; Xing, B.; Loh, T.-P. Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins. Angew. Chem., Int. Ed. 2014, 53 (29), 7491– 7494, DOI: 10.1002/anie.201403121[Crossref], [CAS], Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXovVKltb0%253D&md5=bd00e8d70e768cf9a4b84422b2ee10edAllenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and ProteinsAbbas, Ata; Xing, Bengang; Loh, Teck-PengAngewandte Chemie, International Edition (2014), 53 (29), 7491-7494CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)In this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aq. medium with excellent selectivity and quant. conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biol. systems. Addnl., fluorescent-labeling studies demonstrated that the installation of a C-terminal allenamide moiety onto various mols. of interest may supply a new methodol. towards the site-specific labeling of cysteine-contg. proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.
- 76Pedzisa, L.; Li, X.; Rader, C.; Roush, W. R. Assessment of Reagents for Selenocysteine Conjugation and the Stability of Selenocysteine Adducts. Org. Biomol. Chem. 2016, 14 (22), 5141– 5147, DOI: 10.1039/C6OB00775A[Crossref], [PubMed], [CAS], Google Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XotF2msL4%253D&md5=3f96465dfc64746df2ef4704ad767dc9Assessment of reagents for selenocysteine conjugation and the stability of selenocysteine adductsPedzisa, Lee; Li, Xiuling; Rader, Christoph; Roush, William R.Organic & Biomolecular Chemistry (2016), 14 (22), 5141-5147CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Conventional antibody-drug conjugates (ADCs) are heterogeneous mixts. that have poor pharmacokinetic properties and decreased efficacy relative to homogenous ADCs. Furthermore, ADCs that are maleimide-based often have inadequate circulatory stability, which can result in premature drug release with consequent off-target toxicities. Selenocysteine-modified antibodies have been developed that allow site-specific antibody conjugation, yielding homogeneous ADCs. Herein, we survey several electrophilic functional groups that react with selenocystine with high efficiency. Several of these result in conjugates with stabilities that are superior to maleimide conjugates. Among these, the allenamide functional group reacts with notably high efficiency, leads to conjugates with remarkable stability, and shows exquisite selectivity for selenocysteine conjugation.
- 77Chen, D.; Guo, D.; Yan, Z.; Zhao, Y. Allenamide as a Bioisostere of Acrylamide in the Design and Synthesis of Targeted Covalent Inhibitors. MedChemComm 2018, 9 (2), 244– 253, DOI: 10.1039/C7MD00571G[Crossref], [PubMed], [CAS], Google Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFGns7rO&md5=919f67c6ee52d1996f0e5ccdfbe77279Allenamide as a bioisostere of acrylamide in the design and synthesis of targeted covalent inhibitorsChen, Deheng; Guo, Dexiang; Yan, Ziqin; Zhao, YujunMedChemComm (2018), 9 (2), 244-253CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)The success of acrylamide-contg. drugs in treating cancers has spurred a passion to search for acrylamide bioisosteres. In our endeavour, we have identified that an allenamide group can be a reactive bioisostere of the acrylamide group. In our development of allenamide-contg. compds., we found that the most potent compd., 14, inhibited the kinase activities of both T790M/L858R double mutant and wild type EGFR in a low nM range. 14 also inhibited the growth of NCI-H1975 lung cancer cells at IC50 = 33 nM, which is comparable to that of acrylamide-contg. osimertinib. The western blot anal. showed that the phosphorylation of EGFR, AKT, and ERK1/2 was simultaneously inhibited in a dose-dependent manner when NCI-H1975 cells were treated with 14. By measuring the conjugate addn. product formed by 14 and GSH, we obtained a reaction rate const. of 302.5 × 10-3 min-1, which is about 30-fold higher than that of osimertinib. Taken together, our data suggest that the allenamide-contg. compds. inhibited EGFR kinases through covalent modifications. Our study indicates that the allenamide group could serve as an alternative electrophilic warhead in the design of targeted covalent inhibitors, and this bioisostere replacement may have broad applications in medicinal chem.
- 78Awoonor-Williams, E.; Rowley, C. N. How Reactive Are Druggable Cysteines in Protein Kinases?. J. Chem. Inf. Model. 2018, 58 (9), 1935– 1946, DOI: 10.1021/acs.jcim.8b00454[ACS Full Text], [CAS], Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFejsLrN&md5=9ebb19e671ee3a29af129733d5803544How Reactive are Druggable Cysteines in Protein Kinases?Awoonor-Williams, Ernest; Rowley, Christopher N.Journal of Chemical Information and Modeling (2018), 58 (9), 1935-1946CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)Targeted covalent-inhibitors (TCIs) have been successfully developed as high-affinity and selective inhibitors of enzymes of the protein kinase family. These drugs typically act by undergoing an electrophilic addn. with an active site cysteine residue, so design of a TCI begins with the identification of a "druggable" cysteine. These electrophilic addns. generally require the deprotonation of the thiol to form a reactive anionic thiolate, so the acidity of the residue is a crit. factor. Few exptl. measurements of the pKa's of druggable cysteines have been reported, so computational prediction could prove to be very important in selecting reactive cysteine targets. Here, we report the computed pKa's of druggable cysteines in select protein kinases which are of clin. relevance for targeted therapies. The pKa's of the cysteines were calcd. using advanced computational methods based on all-atom replica-exchange thermodn. integration mol. dynamics simulations in explicit solvent. We found that the acidities of druggable cysteines within protein kinases are diverse and elevated, indicating enormous differences in their reactivity. Const. pH mol. dynamics simulations were also performed on select protein kinases, with results confirming this varied range in the acidities of druggable cysteines. Many of these active site cysteines have low exposure to solvent mols., elevating their pKa. Electrostatic interactions with nearby anionic residues also elevate the pKa's of cysteine residues in the active site. The results suggest that some cysteine residues within kinase binding sites will be slow to react with a TCI due to their low acidity. Several oncogenic kinase mutations were also modeled and found to have similar pKa's to the wild-type.
- 79Koniev, O.; Leriche, G.; Nothisen, M.; Remy, J.-S.; Strub, J.-M.; Schaeffer-Reiss, C.; Van Dorsselaer, A.; Baati, R.; Wagner, A. Selective Irreversible Chemical Tagging of Cysteine with 3-Arylpropiolonitriles. Bioconjugate Chem. 2014, 25 (2), 202– 206, DOI: 10.1021/bc400469d[ACS Full Text], [CAS], Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtl2nsQ%253D%253D&md5=6a55f6f7c3ca774016d6c92c5e21812cSelective Irreversible Chemical Tagging of Cysteine with 3-ArylpropiolonitrilesKoniev, Oleksandr; Leriche, Geoffray; Nothisen, Marc; Remy, Jean-Serge; Strub, Jean-Marc; Schaeffer-Reiss, Christine; Van Dorsselaer, Alain; Baati, Rachid; Wagner, AlainBioconjugate Chemistry (2014), 25 (2), 202-206CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Exquisite chemoselectivity for cysteine has been found for a novel class of remarkably hydrolytically stable reagents, 3-arylpropiolonitriles (APN). The efficacy of the APN-mediated tagging was benchmarked against other cysteine-selective methodologies in a model study on a series of traceable amino acid derivs. The selectivity of the methodol. was further explored on peptide mixts. obtained by trypsin digestion of lysozyme. Addnl., the superior stability of APN-cysteine conjugates in aq. media, human plasma, and living cells makes this new thiol-click reaction a promising methodol. for applications in bioconjugation.
- 80Shiu, H.-Y.; Chan, T.-C.; Ho, C.-M.; Liu, Y.; Wong, M.-K.; Che, C.-M. Electron-Deficient Alkynes as Cleavable Reagents for the Modification of Cysteine-Containing Peptides in Aqueous Medium. Chem. - Eur. J. 2009, 15 (15), 3839– 3850, DOI: 10.1002/chem.200800669[Crossref], [PubMed], [CAS], Google Scholar80https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksleitr0%253D&md5=cdf4acf18e4737c673a807ed1968aae6Electron-deficient alkynes as cleavable reagents for the modification of cysteine-containing peptides in aqueous mediumShiu, Hoi-Yan; Chan, Tak-Chung; Ho, Chi-Ming; Liu, Yungen; Wong, Man-Kin; Che, Chi-MingChemistry - A European Journal (2009), 15 (15), 3839-3850CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)An efficient method has been developed for the chemoselective cysteine modification of unprotected peptides and proteins in aq. media through the formation of a vinyl sulfide linkage by using electron-deficient alkynes, including alkynoic amides, esters and alky-nones. The terminal alky-none-modified peptides could be converted back into the unmodified peptides (81% isolated yield) by adding thiols under mild conditions. The usefulness of this thiol-assisted cleavage of the vinyl sulfide linkage in peptides has been exemplified by the enrichment of a cysteine-contg. peptide (71% recovery) from a mixt. of cysteine-contg. and non-cysteine-contg. peptides.
- 81Friedman, M.; Wall, J. S. Additive Linear Free-Energy Relationships in Reaction Kinetics of Amino Groups with α,β-Unsaturated Compounds. J. Org. Chem. 1966, 31 (9), 2888– 2894, DOI: 10.1021/jo01347a036[ACS Full Text], [CAS], Google Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF28Xkslert7k%253D&md5=478d318c5d9ffb47182f119daf52fac3Additive linear free-energy relationships in reaction kinetics of amino groups with α,β-unsaturated compoundsFriedman, Mendel; Wall, Joseph S.Journal of Organic Chemistry (1966), 31 (9), 2888-94CODEN: JOCEAH; ISSN:0022-3263.Reaction rates of nucleophilic addns. of amino groups in amino acids and peptides to α,β-unsatd. compds. were studied as functions of structural variations in both reactants. The obsd. wide variations in rates are correlated by a linear free-energy equation, which includes parameters assocd. with polar and steric factors of both reactants. This equation has theor. significance and should be of value for the calcn. of predicted polar and steric factors and reaction rates of a large no. of compds. Linear relationships were obsd. between reaction rates for a no. of vinyl compds. and several physicochem. parameters inherent in structural features assocd. with these compds. These relationships indicate that resonance stabilization of ground and transition rates appears to be the main factor that governs relative electrophilic reactivities of α,β-unsatd. compds. 25 refs.
- 82Cavins, J. F.; Friedman, M. An Internal Standard for Amino Acid Analyses: S-β-(4-Pyridylethyl)-l-Cysteine. Anal. Biochem. 1970, 35 (2), 489– 493, DOI: 10.1016/0003-2697(70)90211-3[Crossref], [PubMed], [CAS], Google Scholar82https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3cXktlWgu7s%253D&md5=e2b5a8763c785a40223664b09ef4dc85Reactions of amino acids, peptides, and proteins with α,β-unsaturated compounds. XIII. Internal standard for amino acid analyses: S-β-(4-pyridylethyl)-L-cysteineCavins, James F.; Friedman, MendelAnalytical Biochemistry (1970), 35 (2), 489-93CODEN: ANBCA2; ISSN:0003-2697.The new amino acid, S-β-(4-pyridylethyl)-L-cysteine (I), was prepd. by treating L-cysteine with 4-vinylpyridine in an aq. medium containing Et3N. The postulated structure for I was confirmed by IR, NMR, and mass spectroscopic analyses. I is stable to acid under conditions used for protein hydrolysis, it elutes on a basic column as a discrete peak before arginine, and its ninhydrin color is linear with concn. The new amino acid has been evaluated as an internal std. for amino acid analyses. Equally excellent results were obtained when I was added to the protein either before or after hydrolysis. Addn. of I before hydrolysis eliminates the need for N anal. of the hydrolyzate and for accurate sample application to the column.
- 83Gill, A. L.; Frederickson, M.; Cleasby, A.; Woodhead, S. J.; Carr, M. G.; Woodhead, A. J.; Walker, M. T.; Congreve, M. S.; Devine, L. A.; Tisi, D.; O’Reilly, M.; Seavers, L. C. A.; Davis, D. J.; Curry, J.; Anthony, R.; Padova, A.; Murray, C. W.; Carr, R. A. E.; Jhoti, H. Identification of Novel P38α MAP Kinase Inhibitors Using Fragment-Based Lead Generation. J. Med. Chem. 2005, 48 (2), 414– 426, DOI: 10.1021/jm049575n[ACS Full Text], [CAS], Google Scholar83https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtFCiu7nJ&md5=a128859d7f3725f2672512331b6bdc83Identification of Novel p38α MAP Kinase Inhibitors Using Fragment-Based Lead GenerationGill, Adrian L.; Frederickson, Martyn; Cleasby, Anne; Woodhead, Steven J.; Carr, Maria G.; Woodhead, Andrew J.; Walker, Margaret T.; Congreve, Miles S.; Devine, Lindsay A.; Tisi, Dominic; O'Reilly, Marc; Seavers, Lisa C. A.; Davis, Deborah J.; Curry, Jayne; Anthony, Rachel; Padova, Alessandro; Murray, Christopher W.; Carr, Robin A. E.; Jhoti, HarrenJournal of Medicinal Chemistry (2005), 48 (2), 414-426CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We describe the structure-guided optimization of the mol. fragments 2-amino-3-benzyloxypyridine 1 (IC50 1.3 mM) and 3-(2-(4-pyridyl)ethyl)indole 2 (IC50 35 μM) identified using X-ray crystallog. screening of p38α MAP kinase. Using two sep. case studies, the article focuses on the key compds. synthesized, the structure-activity relationships and the binding mode observations made during this optimization process, resulting in two potent lead series that demonstrate significant increases in activity. We describe the process of compd. elaboration either through the growing out from fragments into adjacent pockets or through the conjoining of overlapping fragments and demonstrate that we have exploited the mobile conserved activation loop, consisting in part of Asp168-Phe169-Gly170 (DFG), to generate significant improvements in potency and kinase selectivity.
- 84Raux, E.; Klenc, J.; Blake, A.; Sączewski, J.; Strekowski, L. Conjugate Addition of Nucleophiles to the Vinyl Function of 2-Chloro-4-Vinylpyrimidine Derivatives. Molecules 2010, 15 (3), 1973– 1984, DOI: 10.3390/molecules15031973[Crossref], [PubMed], [CAS], Google Scholar84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktVymu7s%253D&md5=7795a89f928d41ec009dd2202f7f2a60Conjugate addition of nucleophiles to the vinyl function of 2-chloro-4-vinylpyrimidine derivativesRaux, Elizabeth; Klenc, Jeffrey; Blake, Ava; Saczewski, Jaroslaw; Strekowski, LucjanMolecules (2010), 15 (), 1973-1984CODEN: MOLEFW; ISSN:1420-3049. (Molecular Diversity Preservation International)Conjugate addn. reaction of various nucleophiles across the vinyl group of 2-chloro-4-vinylpyrimidine, 2-chloro-4-(1-phenylvinyl)pyrimidine and 2-chloro-4-vinylquinazoline provides the corresponding 2-chloro-4-(2-substituted ethyl)pyrimidines and 2-chloro-4-(2-substituted ethyl)quinazolines. Treatment of these products, without isolation, with N-methylpiperazine results in nucleophilic displacement of chloride and yields the corresponding 2,4-disubstituted pyrimidines, e.g., I and quinazolines, e.g., II.
- 85Burns, A. R.; Kerr, J. H.; Kerr, W. J.; Passmore, J.; Paterson, L. C.; Watson, A. J. B. Tuned Methods for Conjugate Addition to a Vinyl Oxadiazole; Synthesis of Pharmaceutically Important Motifs. Org. Biomol. Chem. 2010, 8 (12), 2777– 2783, DOI: 10.1039/c001772h[Crossref], [PubMed], [CAS], Google Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmvVWktL4%253D&md5=9a7a8ec8555b6c165d9f2eaa6c1eb8b5Tuned methods for conjugate addition to a vinyl oxadiazole; synthesis of pharmaceutically important motifsBurns, Alan R.; Kerr, Jennifer H.; Kerr, William J.; Passmore, Joanna; Paterson, Laura C.; Watson, Allan J. B.Organic & Biomolecular Chemistry (2010), 8 (12), 2777-2783CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)The addn. of various nucleophiles to 3-phenyl-5-vinyl-1,2,4-oxadiazole is described. Following optimization, individual protocols tuned for the use of each specific class of reagent have been developed to allow the installation of nitrogen, sulfur, oxygen, and carbon nucleophiles, and leading to the prepn. of a series of compds. contg. the pharmaceutically important oxadiazole motif.
- 86Kuchař, M.; Hocek, M.; Pohl, R.; Votruba, I.; Shih, I.; Mabery, E.; Mackman, R. Synthesis, Cytostatic, and Antiviral Activity of Novel 6-[2-(Dialkylamino)Ethyl]-, 6-(2-Alkoxyethyl)-, 6-[2-(Alkylsulfanyl)Ethyl]-, and 6-[2-(Dialkylamino)Vinyl]Purine Nucleosides. Bioorg. Med. Chem. 2008, 16 (3), 1400– 1424, DOI: 10.1016/j.bmc.2007.10.063[Crossref], [PubMed], [CAS], Google Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhvVWgsr8%253D&md5=1b4eaa340d0f2abc73bbbe2530b7ebddSynthesis, cytostatic, and antiviral activity of novel 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, 6-[2-(alkylsulfanyl)ethyl]-, and 6-[2-(dialkylamino)vinyl]purine nucleosidesKuchar, Martin; Hocek, Michal; Pohl, Radek; Votruba, Ivan; Shih, I-hung; Mabery, Eric; Mackman, RichardBioorganic & Medicinal Chemistry (2008), 16 (3), 1400-1424CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)An efficient and facile synthesis of a large series of diverse 6-[2-(dialkylamino)vinyl]-, 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, and 6-[2-(alkylsulfanyl)ethyl]purine nucleosides (35 examples of both ribo- and 2'-deoxyribonucleosides) was developed. The key transformations involved conjugate nucleophilic addns. of amines, alcoholates, or thiolates to Tol-protected 6-alkylylpurine or 6-vinylpurine nucleosides. 6-[(2-Dialkylamino)vinyl]- and some 6-[(2-dialkylamino)ethyl]purine ribonucleosides exerted significant cytostatic effects and some anti-HCV activity with low selectivity.
- 87Il’yasov, E. A.; Galust’yan, G. G. Homolytic Addition of 1-Alkanethiols to 5-Ethynyl-2-Methylpyridine. Chem. Heterocycl. Compd. 1999, 35 (10), 1187– 1189, DOI: 10.1007/BF02323377
- 88Wipf, P.; Graham, T. H. Synthesis and Hetero-Michael Addition Reactions of 2-Alkynyl Oxazoles and Oxazolines. Org. Biomol. Chem. 2005, 3 (1), 31– 35, DOI: 10.1039/b413604g[Crossref], [PubMed], [CAS], Google Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVylt7vE&md5=861c6c74059a0ee8a8667ea89646a201Synthesis and hetero-Michael addition reactions of 2-alkynyl oxazoles and oxazolinesWipf, Peter; Graham, Thomas H.Organic & Biomolecular Chemistry (2005), 3 (1), 31-35CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Stereoselective conjugate addns. of alcs., amines, thiols, and halides to C(2)-alkynyl oxazoles and oxazolines provide a versatile entry to heterocyclic building blocks. For example, the reaction of ethanethiol with 2-(ethynyl)-4-oxazolecarboxylic acid Me ester (I) gave 2-[(1Z)-2-(ethylthio)ethenyl]-4-oxazolecarboxylic acid Me ester (II) stereoselectively.
- 89Li, Q.-F.; Yang, Y.; Maleckis, A.; Otting, G.; Su, X.-C. Thiol–Ene Reaction: A Versatile Tool in Site-Specific Labelling of Proteins with Chemically Inert Tags for Paramagnetic NMR. Chem. Commun. 2012, 48 (21), 2704– 2706, DOI: 10.1039/c2cc17900h[Crossref], [PubMed], [CAS], Google Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XitFCgurw%253D&md5=bd028717e4192f1d5f35a96892988d7cThiol-ene reaction: a versatile tool in site-specific labelling of proteins with chemically inert tags for paramagnetic NMRLi, Qing-Feng; Yang, Yin; Maleckis, Ansis; Otting, Gottfried; Su, Xun-ChengChemical Communications (Cambridge, United Kingdom) (2012), 48 (21), 2704-2706CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Site-specific tagging of proteins with paramagnetic lanthanides generates valuable long-range structure restraints for structural biol. by NMR spectroscopy. The authors show that the thiol-ene addn. reaction offers a powerful tool for tagging proteins in a chem. stable manner with very small lanthanide tags.
- 90Yang, Y.; Li, Q.-F.; Cao, C.; Huang, F.; Su, X.-C. Site-Specific Labeling of Proteins with a Chemically Stable, High-Affinity Tag for Protein Study. Chem. - Eur. J. 2013, 19 (3), 1097– 1103, DOI: 10.1002/chem.201202495[Crossref], [PubMed], [CAS], Google Scholar90https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1OgsLrM&md5=6bab43146e47348dc0ed1a1d0fa6c718Site-Specific Labeling of Proteins with a Chemically Stable, High-Affinity Tag for Protein StudyYang, Yin; Li, Qing-Feng; Cao, Chan; Huang, Feng; Su, Xun-ChengChemistry - A European Journal (2013), 19 (3), 1097-1103CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)Site-specific labeling of proteins with paramagnetic lanthanides offers unique opportunities by virtue of NMR spectroscopy in structural biol. In particular, these paramagnetic data, generated by the anisotropic paramagnetism including pseudocontact shifts (PCS), residual dipolar couplings (RDC), and paramagnetic relaxation enhancement (PRE), are highly valuable in structure detn. and mobility studies of proteins and protein-ligand complexes. Herein, we present a new way to label proteins in a site-specific manner with a high-affinity and chem. stable tag, 4-vinyl(pyridine-2,6-diyl)bismethylenenitrilo tetrakis(acetic acid) (4VPyMTA), through thiol alkylation. Its performance has been demonstrated in G47C and E64C mutants of human ubiquitin both in vitro and in a crowded environment. In comparison with the published tags, 4VPyMTA has several interesting features: (1) it has a very high binding affinity for lanthanides (higher than EDTA), (2) there is no heterogeneity in complexes with lanthanides, (3) the derivatized protein is stable and potentially applicable to the in situ anal. of proteins.
- 91Ma, F.-H.; Chen, J.-L.; Li, Q.-F.; Zuo, H.-H.; Huang, F.; Su, X.-C. Kinetic Assay of the Michael Addition-Like Thiol–Ene Reaction and Insight into Protein Bioconjugation. Chem. - Asian J. 2014, 9 (7), 1808– 1816, DOI: 10.1002/asia.201402095[Crossref], [PubMed], [CAS], Google Scholar91https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXosV2gsro%253D&md5=e4878dfc5437061ec2b42f5098341589Kinetic Assay of the Michael Addition-Like Thiol-Ene Reaction and Insight into Protein BioconjugationMa, Fei-He; Chen, Jia-Liang; Li, Qing-Feng; Zuo, Hui-Hui; Huang, Feng; Su, Xun-ChengChemistry - An Asian Journal (2014), 9 (7), 1808-1816CODEN: CAAJBI; ISSN:1861-4728. (Wiley-VCH Verlag GmbH & Co. KGaA)The chem. modification of proteins is a valuable technique in understanding the functions, interactions, and dynamics of proteins. Reactivity and selectivity are key issues in current chem. modification of proteins. The Michael addn.-like thiol-ene reaction is a useful tool that can be used to tag proteins with high selectivity for the solvent-exposed thiol groups of proteins. To obtain insight into the bioconjugation of proteins with this method, a kinetic anal. was performed. New vinyl-substituted pyridine derivs. were designed and synthesized. The reactivity of these vinyl tags with L-cysteine was evaluated by UV absorption and high-resoln. NMR spectroscopy. The results show that protonation of pyridine plays a key role in the overall reaction rates. The kinetic parameters were assessed in protein modification. The different reactivities of these vinyl tags with solvent-exposed cysteine is valuable information in the selective labeling of proteins with multiple functional groups.
- 92Wood, E. R.; Shewchuk, L. M.; Ellis, B.; Brignola, P.; Brashear, R. L.; Caferro, T. R.; Dickerson, S. H.; Dickson, H. D.; Donaldson, K. H.; Gaul, M.; Griffin, R. J.; Hassell, A. M.; Keith, B.; Mullin, R.; Petrov, K. G.; Reno, M. J.; Rusnak, D. W.; Tadepalli, S. M.; Ulrich, J. C.; Wagner, C. D.; Vanderwall, D. E.; Waterson, A. G.; Williams, J. D.; White, W. L.; Uehling, D. E. 6-Ethynylthieno[3,2-d]- and 6-Ethynylthieno[2,3-d]Pyrimidin-4-Anilines as Tunable Covalent Modifiers of ErbB Kinases. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (8), 2773– 2778, DOI: 10.1073/pnas.0708281105[Crossref], [PubMed], [CAS], Google Scholar92https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjtVSjtbw%253D&md5=fbfeeff1f03e8f7fb2c9e228231ff92e6-Ethynylthieno[3,2-d]- and 6-ethynylthieno[2,3-d]pyrimidin-4-anilines as tunable covalent modifiers of ErbB kinasesWood, Edgar R.; Shewchuk, Lisa M.; Ellis, Byron; Brignola, Perry; Brashear, Ronald L.; Caferro, Thomas R.; Dickerson, Scott H.; Dickson, Hamilton D.; Donaldson, Kelly H.; Gaule, Michael; Griffin, Robert J.; Hassell, Anne M.; Keith, Barry; Mullin, Robert; Petrov, Kimberly G.; Reno, Michael J.; Rusnak, David W.; Tadepalli, Sarva M.; Ulrich, John C.; Wagner, Craig D.; Vanderwall, Dana E.; Waterson, Alex G.; Williams, Jon D.; White, Wendy L.; Uehling, David E.Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (8), 2773-2778CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Anal. of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678-1020) with a structurally related propargylic amine (8). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure-activity relation understanding of the covalent modifying potential and biol. activity of a series of acetylenic thienopyrimidine compds. with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.
- 93Smaill, J. B.; Rewcastle, G. W.; Loo, J. A.; Greis, K. D.; Chan, O. H.; Reyner, E. L.; Lipka, E.; Showalter, H. D. H.; Vincent, P. W.; Elliott, W. L.; Denny, W. A. Tyrosine Kinase Inhibitors. 17. Irreversible Inhibitors of the Epidermal Growth Factor Receptor: 4-(Phenylamino)Quinazoline- and 4-(Phenylamino)Pyrido[3,2-d]Pyrimidine-6-Acrylamides Bearing Additional Solubilizing Functions. J. Med. Chem. 2000, 43 (7), 1380– 1397, DOI: 10.1021/jm990482t[ACS Full Text], [CAS], Google Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhs1Crtr0%253D&md5=6c569a822c398df08559abde31a1d4acTyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4-(Phenylamino)quinazoline- and 4-(Phenylamino)pyrido[3,2-d]pyrimidine-6-acrylamides bearing additional solubilizing functionsSmaill, Jeff B.; Rewcastle, Gordon W.; Loo, Joseph A.; Greis, Kenneth D.; Chan, O. Helen; Reyner, Eric L.; Lipka, Elke; Showalter, H. D. Hollis; Vincent, Patrick W.; Elliott, William L.; Denny, William A.Journal of Medicinal Chemistry (2000), 43 (7), 1380-1397CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)4-Anilinoquinazoline- and 4-anilinopyrido[3,2-d]pyrimidine-6-acrylamides substituted with solubilizing 7-alkylamine or 7-alkoxyamine side chains were prepd. by reaction of the corresponding 6-amines with acrylic acid or acrylic acid anhydrides. In the pyrido[3,2-d]pyrimidine series, the intermediate 6-amino-7-alkylamines were prepd. from 7-bromo-6-fluoropyrido[3,2-d]pyrimidine via Stille coupling with the appropriate stannane under palladium(0) catalysis. This proved a versatile method for the introduction of cationic solubilizing side chains. The compds. were evaluated for their inhibition of phosphorylation of the isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells. Quinazoline analogs with 7-alkoxyamine solubilizing groups were potent irreversible inhibitors of the isolated EGFR enzyme, with IC50[app] values from 2 to 4 nM, and potently inhibited both EGFR and erbB2 autophosphorylation in cells. 7-Alkylamino- and 7-alkoxyaminopyrido[3,2-d]pyrimidines were also irreversible inhibitors with equal or superior potency against the isolated enzyme but were less effective in the cellular autophosphorylation assays. Both quinazoline- and pyrido[3,2-d]pyrimidine-6-acrylamides bound at the ATP site alkylating cysteine 773, as shown by electrospray ionization mass spectrometry, and had similar rates of absorptive and secretory transport in Caco-2 cells. A comparison of two 7-propoxymorpholide analogs showed that the pyrido[3,2-d]pyrimidine-6-acrylamide had greater amide instability and higher acrylamide reactivity, being converted to glutathione adducts in cells more rapidly than the corresponding quinazoline. This difference may contribute to the obsd. lower cellular potency of the pyrido[3,2-d]pyrimidine-6-acrylamides. Selected compds. showed high in vivo activity against A431 xenografts on oral dosing, with the quinazolines being superior to the pyrido[3,2-d]pyrimidines. Overall, the quinazolines proved superior to previous analogs in terms of aq. soly., potency, and in vivo antitumor activity, and one example (CI 1033) has been selected for clin. evaluation.
- 94Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.; Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.; Greenberger, L. M.; Wang, Y.-F.; Wissner, A. 6-Substituted-4-(3-Bromophenylamino)Quinazolines as Putative Irreversible Inhibitors of the Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor (HER-2) Tyrosine Kinases with Enhanced Antitumor Activity. J. Med. Chem. 2001, 44 (17), 2719– 2734, DOI: 10.1021/jm0005555[ACS Full Text], [CAS], Google Scholar94https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXltFChsb8%253D&md5=989e6e77195324d92e70ca13f532e1cf6-Substituted-4-(3-bromophenylamino)quinazolines as Putative Irreversible Inhibitors of the Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor (HER-2) Tyrosine Kinases with Enhanced Antitumor ActivityTsou, Hwei-Ru; Mamuya, Nellie; Johnson, Bernard D.; Reich, Marvin F.; Gruber, Brian C.; Ye, Fei; Nilakantan, Ramaswamy; Shen, Ru; Discafani, Carolyn; DeBlanc, Ronald; Davis, Rachel; Koehn, Frank E.; Greenberger, Lee M.; Wang, Yu-Fen; Wissner, AllanJournal of Medicinal Chemistry (2001), 44 (17), 2719-2734CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of new 6-substituted-4-(3-bromophenylamino)quinazoline derivs. that may function as irreversible inhibitors of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases have been prepd. These inhibitors have, at the C-6 position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solubilizing substituents. These compds. were prepd. by acylation of 6-amino-4-(3-bromophenylamino)quinazoline with unsatd. acid chlorides or mixed anhydrides. We show that attaching a basic functional group onto the Michael acceptor results in greater reactivity, due to intramol. catalysis of the Michael addn. and/or an inductive effect of the protonated basic group. This, along with improved water soly., results in compds. with enhanced biol. properties. We present mol. modeling and exptl. evidence that these inhibitors interact covalently with the target enzymes. One compd., (I) was shown to have excellent oral activity in a human epidermoid carcinoma (A431) xenograft model in nude mice.
- 95Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.; Johnson, B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; Shi, X.; Rabindran, S. K.; Gruber, B. C.; Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang, Y.-F.; Greenberger, L. M.; Tsou, H.-R. Synthesis and Structure–Activity Relationships of 6,7-Disubstituted 4-Anilinoquinoline-3-Carbonitriles. The Design of an Orally Active, Irreversible Inhibitor of the Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor (EGFR) and the Human Epidermal Growth Factor Receptor-2 (HER-2). J. Med. Chem. 2003, 46 (1), 49– 63, DOI: 10.1021/jm020241c[ACS Full Text], [CAS], Google Scholar95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XptFOlsbk%253D&md5=db70c2f78c62824810854295d06ef6a4Synthesis and Structure-Activity Relationships of 6,7-Disubstituted 4-Anilinoquinoline-3-carbonitriles. The Design of an Orally Active, Irreversible Inhibitor of the Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor (EGFR) and the Human Epidermal Growth Factor Receptor-2 (HER-2)Wissner, Allan; Overbeek, Elsebe; Reich, Marvin F.; Floyd, M. Brawner; Johnson, Bernard D.; Mamuya, Nellie; Rosfjord, Edward C.; Discafani, Carolyn; Davis, Rachel; Shi, Xiaoqing; Rabindran, Sridhar K.; Gruber, Brian C.; Ye, Fei; Hallett, William A.; Nilakantan, Ramaswamy; Shen, Ru; Wang, Yu-Fen; Greenberger, Lee M.; Tsou, Hwei-RuJournal of Medicinal Chemistry (2003), 46 (1), 49-63CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of 6,7-disubstituted 4-anilinoquinoline-3-carbonitriles, e.g., I, that function as irreversible inhibitors of EGFR and HER-2 kinases have been prepd. These inhibitors have, at the 6-position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solubilizing substituents. These compds. were prepd. by acylation of 6-amino-4-(arylamino)quinoline-3-carbonitriles, e.g., II, with unsatd. acid chlorides or mixed anhydrides. Competitive reactivity studies were performed, showing that attaching a dialkylamino group onto the end of the Michael acceptor results in compds. with greater reactivity due to intramol. catalysis of the Michael addn. This, along with improved water-soly. results in compds. with enhanced biol. properties. Mol. modeling results consistent with the proposed mechanism of inhibition are presented. One compd., I (EKB-569), which shows excellent oral in vivo activity, was selected for further studies and is currently in phase I clin. trials for the treatment of cancer.
- 96Hubbard, R. D.; Dickerson, S. H.; Emerson, H. K.; Griffin, R. J.; Reno, M. J.; Hornberger, K. R.; Rusnak, D. W.; Wood, E. R.; Uehling, D. E.; Waterson, A. G. Dual EGFR/ErbB-2 Inhibitors from Novel Pyrrolidinyl-Acetylenic Thieno[3,2-d]Pyrimidines. Bioorg. Med. Chem. Lett. 2008, 18 (21), 5738– 5740, DOI: 10.1016/j.bmcl.2008.09.090[Crossref], [PubMed], [CAS], Google Scholar96https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht12rsrfI&md5=ce42d0aadfaadd02bc026d781560665fDual EGFR/ErbB-2 inhibitors from novel pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidinesHubbard, Robert D.; Dickerson, Scott H.; Emerson, Holly K.; Griffin, Robert J.; Reno, Michael J.; Hornberger, Keith R.; Rusnak, David W.; Wood, Edgar R.; Uehling, David E.; Waterson, Alex G.Bioorganic & Medicinal Chemistry Letters (2008), 18 (21), 5738-5740CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)A novel class of substituted pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines has been identified that are potent and selective inhibitors of both EGFR/ErbB-2 receptor tyrosine kinases. The inhibitors are found to display a range of enzyme and cellular potency and also to display a varying level of covalent modification of the kinase targets. Selected mols., including compd. (I), were found to be potent in enzymic and cellular assays while also demonstrating exposure in the mouse from an oral dose.
- 97Stevens, K. L.; Alligood, K. J.; Alberti, J. G. B.; Caferro, T. R.; Chamberlain, S. D.; Dickerson, S. H.; Dickson, H. D.; Emerson, H. K.; Griffin, R. J.; Hubbard, R. D.; Keith, B. R.; Mullin, R. J.; Petrov, K. G.; Gerding, R. M.; Reno, M. J.; Rheault, T. R.; Rusnak, D. W.; Sammond, D. M.; Smith, S. C.; Uehling, D. E.; Waterson, A. G.; Wood, E. R. Synthesis and Stereochemical Effects of Pyrrolidinyl-Acetylenic Thieno[3,2-d]Pyrimidines as EGFR and ErbB-2 Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (1), 21– 26, DOI: 10.1016/j.bmcl.2008.11.023[Crossref], [PubMed], [CAS], Google Scholar97https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlvFKl&md5=e9fbe3252cb62022196d8424e1189ff7Synthesis and stereochemical effects of pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines as EGFR and ErbB-2 inhibitorsStevens, Kirk L.; Alligood, Krystal J.; Alberti, Jennifer G. Badiang; Caferro, Thomas R.; Chamberlain, Stanley D.; Dickerson, Scott H.; Dickson, Hamilton D.; Emerson, Holly K.; Griffin, Robert J.; Hubbard, Robert D.; Keith, Barry R.; Mullin, Robert J.; Petrov, Kimberly G.; Gerding, Roseanne M.; Reno, Michael J.; Rheault, Tara R.; Rusnak, David W.; Sammond, Douglas M.; Smith, Stephon C.; Uehling, David E.; Waterson, Alex G.; Wood, Edgar R.Bioorganic & Medicinal Chemistry Letters (2009), 19 (1), 21-26CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)A novel class of pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines, e.g., I, has been identified which potently inhibit the EGFR and ErbB-2 receptor tyrosine kinases. Synthetic modifications of the pyrrolidine carbamate moiety result in a range of effects on enzyme and cellular potency. In addn., the impact of the abs. stereochem. configuration on cellular potency and oral mouse pharmacokinetics is described.
- 98Waterson, A. G.; Petrov, K. G.; Hornberger, K. R.; Hubbard, R. D.; Sammond, D. M.; Smith, S. C.; Dickson, H. D.; Caferro, T. R.; Hinkle, K. W.; Stevens, K. L.; Dickerson, S. H.; Rusnak, D. W.; Spehar, G. M.; Wood, E. R.; Griffin, R. J.; Uehling, D. E. Synthesis and Evaluation of Aniline Headgroups for Alkynyl Thienopyrimidine Dual EGFR/ErbB-2 Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (5), 1332– 1336, DOI: 10.1016/j.bmcl.2009.01.080[Crossref], [PubMed], [CAS], Google Scholar98https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXit1Wms78%253D&md5=7ac528ab1d63f1cd6a6af3985b8034afSynthesis and evaluation of aniline headgroups for alkynyl thienopyrimidine dual EGFR/ErbB-2 kinase inhibitorsWaterson, Alex G.; Petrov, Kimberly G.; Hornberger, Keith R.; Hubbard, Robert D.; Sammond, Douglas M.; Smith, Stephon C.; Dickson, Hamilton D.; Caferro, Thomas R.; Hinkle, Kevin W.; Stevens, Kirk L.; Dickerson, Scott H.; Rusnak, David W.; Spehar, Glenn M.; Wood, Edgar R.; Griffin, Robert J.; Uehling, David E.Bioorganic & Medicinal Chemistry Letters (2009), 19 (5), 1332-1336CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Aniline 'headgroups' were synthesized and incorporated into an alkynyl thienopyrimidine series of EGFR and ErbB-2 inhibitors. Potent inhibition of enzyme activity and cellular proliferation was obsd. In certain instances, protein binding was reduced and oral exposure was found to be somewhat improved relative to compds. contg. the ref. aniline.
- 99Nijmeijer, S.; Engelhardt, H.; Schultes, S.; van de Stolpe, A. C.; Lusink, V.; de Graaf, C.; Wijtmans, M.; Haaksma, E. E. J.; de Esch, I. J. P.; Stachurski, K.; Vischer, H. F.; Leurs, R. Design and Pharmacological Characterization of VUF14480, a Covalent Partial Agonist That Interacts with Cysteine 983.36 of the Human Histamine H4 Receptor. Br. J. Pharmacol. 2013, 170, 89– 100, DOI: 10.1111/bph.12113[Crossref], [PubMed], [CAS], Google Scholar99https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtlSgtbnK&md5=dbaa12c47194793907c66f94dfcbe5fdDesign and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 983.36 of the human histamine H4 receptorNijmeijer, S.; Engelhardt, H.; Schultes, S.; van de Stolpe, A. C.; Lusink, V.; de Graaf, C.; Wijtmans, M.; Haaksma, E. E. J.; de Esch, I. J. P.; Stachurski, K.; Vischer, H. F.; Leurs, R.British Journal of Pharmacology (2013), 170 (1), 89-100CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)Background and Purpose The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E1825.46 in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C983.36 in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C983.36 by functioning as a Michael acceptor. A covalent pyrimidine analog will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research. Exptl. Approach We designed and synthesized VUF14480, and pharmacol. characterized this compd. in hH4 receptor radioligand binding, G protein activation and β-arrestin2 recruitment expts. The ability of VUF14480 to act as a covalent binder was assessed both chem. and pharmacol. Key Results VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and β-arrestin2 recruitment. VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C983.36 prevented this covalent interaction. Conclusion and Implications VUF14480 is thought to bind covalently to the hH4 receptor-C983.36 residue and partially induce hH4 receptor-mediated G protein activation and β-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH4 receptor.
- 100Schapira, A.; Bate, G.; Kirkpatrick, P. Rasagiline. Nat. Rev. Drug Discovery 2005, 4 (8), 625– 626, DOI: 10.1038/nrd1803[Crossref], [PubMed], [CAS], Google Scholar100https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmvVOqt70%253D&md5=d518736530887a0552bc4a2469ddf75cFresh from the pipeline: RasagilineSchapira, Anthony; Bate, Guy; Kirkpatrick, PeterNature Reviews Drug Discovery (2005), 4 (8), 625-626CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)There is no expanded citation for this reference.
- 101Youdim, M. B. H.; Gross, A.; Finberg, J. P. M. Rasagiline [N-Propargyl-1R(+)-Aminoindan], a Selective and Potent Inhibitor of Mitochondrial Monoamine Oxidase B. Br. J. Pharmacol. 2001, 132 (2), 500– 506, DOI: 10.1038/sj.bjp.0703826[Crossref], [PubMed], [CAS], Google Scholar101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXht1Sgu7o%253D&md5=692cfb1e7f679e073f6cbdb2c8650ba9Rasagiline [N-propargyl-1R(+)-aminoindan], a selective and potent inhibitor of mitochondrial monoamine oxidase BYoudim, Moussa B. H.; Gross, Aviva; Finberg, John P. M.British Journal of Pharmacology (2001), 132 (2), 500-506CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)Rasagiline [N-propargyl-1R(+)-aminoindan], was examd. for its monoamine oxidase (MAO) A and B inhibitor activities in rats together with its S(-)-enantiomer (TVP 1022) and the racemic compd. (AGN-1135) and compared to selegiline (1-deprenyl). The tissues that were studied for MAO inhibition were the brain, liver and small intestine. While rasagiline and AGN1135 are highly potent selective irreversible inhibitors of MAO in vitro and in vivo, the S(-) enantiomer is relatively inactive in the tissues examd. The in vitro IC50 values for inhibition of rat brain MAO activity by rasagiline are 4.43±0.92 nM (type B), and 412±123 nM (type A). The ED50 values for ex vivo inhibition of MAO in the brain and liver by a single dose of rasagiline are 0.1±0.01, 0.042±0.0045 mg kg-1 resp. for MAO-B, and 6.48±0.81, 2.38±0.35 mg kg-1 resp. for MAO-A. Selective MAO-B inhibition in the liver and brain was maintained on chronic (21 days) oral dosage with ED50 values of 0.014±0.002 and 0.013±0.001 mg kg-1 resp. The degree of selectivity of rasagiline for inhibition of MAO-B as opposed to MAO-A was similar to that of selegiline. Rasagiline was three to 15 times more potent than selegiline for inhibition of MAO-B in rat brain and liver in vivo on acute and chronic administration, but had similar potency in vitro. These data together with lack of tyramine sympathomimetic potentiation by rasagiline, at selective MAO-B inhibitory dosage, indicate that this inhibitor like selegiline may be a useful agent in the treatment of Parkinson's disease in either symptomatic or L-DOPA adjunct therapy, but lack of amphetamine-like metabolites could present a therapeutic advantage for rasagiline.
- 102Wright, A. T.; Song, J. D.; Cravatt, B. F. A Suite of Activity-Based Probes for Human Cytochrome P450 Enzymes. J. Am. Chem. Soc. 2009, 131 (30), 10692– 10700, DOI: 10.1021/ja9037609[ACS Full Text], [CAS], Google Scholar102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXot1ejsbY%253D&md5=73f0ebac489c8df2ad9cc184819f2e43A Suite of Activity-Based Probes for Human Cytochrome P450 EnzymesWright, Aaron T.; Song, Joongyu D.; Cravatt, Benjamin F.Journal of the American Chemical Society (2009), 131 (30), 10692-10700CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Cytochrome P 450 enzymes regulate a variety of endogenous signaling mols. and play central roles in the metab. of xenobiotics and drugs. We recently showed that an aryl alkyne serves as an effective activity-based probe for profiling mouse liver microsomal P450s in vitro and in vivo. However, individual P450s display distinct substrate and inhibitor specificities, indicating that multiple probe structures may be required to achieve comprehensive coverage of this large and diverse enzyme family. Here, we have synthesized a suite of P 450-directed, activity-based protein profiling (ABPP) probes that contain: (1) varied chem. architectures validated as mechanism-based inhibitors of the P 450 enzyme family, and (2) terminal alkyne groups for click chem. conjugation of reporter tags. This set of probes was screened against a wide cross-section of human P450s, leading to the discovery of an optimal set of probes that provide broad coverage of this enzyme family. We used these probes to profile the effects on P 450 activity of aromatase inhibitors in current clin. use for the treatment of breast cancer. We describe the surprising discovery that one of these aromatase inhibitors, anastrozole, significantly increases probe-labeling of P 450 1A2, indicative of a heterotypic cooperativity effect on a central P 450 isoenzyme involved in metabolizing numerous drugs and xenobiotics. The results presented herein greatly expand the suite of ABPP probes for profiling P450s and illuminate new applications for these tools to understand P 450-drug interactions.
- 103Wright, A. T.; Cravatt, B. F. Chemical Proteomic Probes for Profiling Cytochrome P450 Activities and Drug Interactions In Vivo. Chem. Biol. 2007, 14 (9), 1043– 1051, DOI: 10.1016/j.chembiol.2007.08.008[Crossref], [PubMed], [CAS], Google Scholar103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVOnsr7L&md5=a35bc551c4a2d868719fa6710e86d79eChemical Proteomic Probes for Profiling Cytochrome P450 Activities and Drug Interactions In VivoWright, Aaron T.; Cravatt, Benjamin F.Chemistry & Biology (Cambridge, MA, United States) (2007), 14 (9), 1043-1051CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)The cytochrome P 450 superfamily metabolizes many endogenous signaling mols. and drugs. P 450 enzymes are regulated by posttranslational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here we describe a chem. proteomic strategy to profile P 450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a "clickable" handle provided an activity-based probe that labels multiple P 450s both in proteomic exts. and in vivo. This probe was used to record alterations in liver P 450 activities triggered by chem. agents, including inducers of P 450 expression and direct P 450 inhibitors. The chem. proteomic strategy described herein thus offers a versatile method to monitor P 450 activities and small-mol. interactions in any biol. system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class.
- 104van Geel, R.; Pruijn, G. J. M.; van Delft, F. L.; Boelens, W. C. Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide–Alkyne Cycloaddition. Bioconjugate Chem. 2012, 23 (3), 392– 398, DOI: 10.1021/bc200365k[ACS Full Text], [CAS], Google Scholar104https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtVWhsL0%253D&md5=091ab818d0b4582a4377e6a6a305a598Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide-Alkyne Cycloadditionvan Geel, Remon; Pruijn, Ger J. M.; van Delft, Floris L.; Boelens, Wilbert C.Bioconjugate Chemistry (2012), 23 (3), 392-398CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The 1,3-dipolar cycloaddn. of azides with ring-strained alkynes is one of the few bioorthogonal reactions suitable for specific biomol. labeling in complex biol. systems. Nevertheless, azide-independent labeling of proteins by strained alkynes can occur to a varying extent, thereby limiting the sensitivity of assays based on strain-promoted azide-alkyne cycloaddn. (SPAAC). A subset of three cyclooctynes, dibenzocyclooctyne (DIBO), azadibenzocyclooctyne (DIBAC), and bicyclo[6.1.0]nonyne (BCN), was used to evaluate the azide-independent labeling of proteins in vitro. For all three cyclooctynes, thiol-yne addn. with reduced peptidylcysteines is responsible for most of the azide-independent polypeptide labeling. The identity of the reaction product was confirmed by LC-MS and NMR anal. Moreover, undesired thiol-yne reactions can be prevented by alkylating peptidylcysteine thiols with iodoacetamide (IAM). Since IAM is compatible with SPAAC, a more specific azide-dependent labeling is achieved by preincubating proteins contg. reduced cysteines with IAM.
- 105Tian, H.; Sakmar, T. P.; Huber, T. A Simple Method for Enhancing the Bioorthogonality of Cyclooctyne Reagent. Chem. Commun. 2016, 52 (31), 5451– 5454, DOI: 10.1039/C6CC01321J[Crossref], [PubMed], [CAS], Google Scholar105https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XksVaju7k%253D&md5=cc470fc660442717f7286fc528a79aedA simple method for enhancing the bioorthogonality of cyclooctyne reagentTian, He; Sakmar, Thomas P.; Huber, ThomasChemical Communications (Cambridge, United Kingdom) (2016), 52 (31), 5451-5454CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)The cross-reactivity between some cyclooctynes and thiols limits the bioorthogonality of the strain-promoted azide-alkyne cycloaddn. reaction. A low concn. of β-mercaptoethanol significantly reduces the undesirable side reaction between bicyclononyne (BCN) and cysteine and while preserving free cysteines. The authors site-specifically label a genetically encoded azido group in the visual photoreceptor rhodopsin to demonstrate the utility of the strategy.
- 106Haldón, E.; Nicasio, M. C.; Pérez, P. J. Copper-Catalysed Azide–Alkyne Cycloadditions (CuAAC): An Update. Org. Biomol. Chem. 2015, 13 (37), 9528– 9550, DOI: 10.1039/C5OB01457C[Crossref], [PubMed], [CAS], Google Scholar106https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1KmtbrE&md5=829bd0ca7fcb535afde89f531f5e3f1fCopper-catalysed azide-alkyne cycloadditions (CuAAC): an updateHaldon, Estela; Nicasio, M. Carmen; Perez, Pedro J.Organic & Biomolecular Chemistry (2015), 13 (37), 9528-9550CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)A review. The reactions of org. azides and alkynes catalyzed by copper species represent the prototypical examples of click chem were discussed in detailed. The so-called CuAAC reaction (copper-catalyzed azide-alkyne cycloaddn.), discovered in 2002, has been expanded since then to become an excellent tool in org synthesis. In this contribution the recent results described in the literature since 2010 were reviewed, classified according to the nature of the catalyst precursor: copper(I) or copper(II) salts or complexes, metallic or nano-particulated copper and several solid-supported copper systems.
- 107Ekkebus, R.; van Kasteren, S. I.; Kulathu, Y.; Scholten, A.; Berlin, I.; Geurink, P. P.; de Jong, A.; Goerdayal, S.; Neefjes, J.; Heck, A. J. R.; Komander, D.; Ovaa, H. On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases. J. Am. Chem. Soc. 2013, 135 (8), 2867– 2870, DOI: 10.1021/ja309802n[ACS Full Text], [CAS], Google Scholar107https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXit1Sqtbc%253D&md5=d0f813975b6dae3de723facd0b78e47fOn Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in ProteasesEkkebus, Reggy; van Kasteren, Sander I.; Kulathu, Yogesh; Scholten, Arjen; Berlin, Ilana; Geurink, Paul P.; de Jong, Annemieke; Goerdayal, Soenita; Neefjes, Jacques; Heck, Albert J. R.; Komander, David; Ovaa, HuibJournal of the American Chemical Society (2013), 135 (8), 2867-2870CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Active-site directed probes are powerful in studies of enzymic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of deubiquitinating enzymes (DUBs) was obsd. The resulting product was shown to be a quaternary vinyl thioether, as detd. by X-ray crystallog. Proteomic anal. of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward deubiquitinating enzymes. The obsd. reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.
- 108Sommer, S.; Weikart, N. D.; Linne, U.; Mootz, H. D. Covalent Inhibition of SUMO and Ubiquitin-Specific Cysteine Proteases by an in Situ Thiol–Alkyne Addition. Bioorg. Med. Chem. 2013, 21 (9), 2511– 2517, DOI: 10.1016/j.bmc.2013.02.039[Crossref], [PubMed], [CAS], Google Scholar108https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXksFOrt7w%253D&md5=05fcb05ee930d580e1b2d37bbc5e02ccCovalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol-alkyne additionSommer, Stefanie; Weikart, Nadine D.; Linne, Uwe; Mootz, Henning D.Bioorganic & Medicinal Chemistry (2013), 21 (9), 2511-2517CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)Posttranslational modification of proteins with ubiquitin and ubiquitin-like modifiers such as SUMO can be reverted by specific proteases, also referred to as deubiquitinases and isopeptidases, most of which are cysteine-dependent. We have found that the replacement of the conserved C-terminal glycine with propargylamine converts SUMO and ubiquitin to highly efficient covalent inhibitors of their cognate cysteine proteases. Attack of the catalytic cysteine onto the terminal alkyne results in the formation of a vinyl sulfide linkage. Although this reaction is reminiscent of the inhibitory mechanism of the isosteric nitrile inhibitors it was unexpected due to the low electrophilicity of the alkyne group. We show that a precise location of the functional group in the active site of the protease is crucial for the reaction, which was not inhibited by the presence of a radical scavenger. Furthermore, a mutational study of key catalytic residues in the SUMO-protease Senp1, that is H533A and D550A of the catalytic triad and Q597A as part of the oxyanion hole, revealed that these residues are not required for the obsd. covalent adduct formation. We therefore propose that the reaction is an in situ thiol-alkyne addn. Due to the high chem. inertness of the alkyne moiety the resp. protease inhibitors should be well-suited for cellular and therapeutic applications. In keeping with this idea, selective labeling with propargylated SUMO and Ub probes was obsd. in lysates of cell lines expressing the cognate proteases after transient transfection.
- 109Swatek, K. N.; Aumayr, M.; Pruneda, J. N.; Visser, L. J.; Berryman, S.; Kueck, A. F.; Geurink, P. P.; Ovaa, H.; van Kuppeveld, F. J. M.; Tuthill, T. J.; Skern, T.; Komander, D. Irreversible Inactivation of ISG15 by a Viral Leader Protease Enables Alternative Infection Detection Strategies. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (10), 2371– 2376, DOI: 10.1073/pnas.1710617115[Crossref], [PubMed], [CAS], Google Scholar109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1WjsbbL&md5=06a6e74508456cbca5d3449d5c60974cIrreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategiesSwatek, Kirby N.; Aumayr, Martina; Pruneda, Jonathan N.; Visser, Linda J.; Berryman, Stephen; Kueck, Anja F.; Geurink, Paul P.; Ovaa, Huib; van Kuppeveld, Frank J. M.; Tuthill, Tobias J.; Skern, Tim; Komander, DavidProceedings of the National Academy of Sciences of the United States of America (2018), 115 (10), 2371-2376CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin post-translational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the mol. basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.
- 110Arkona, C.; Rademann, J. Propargyl Amides as Irreversible Inhibitors of Cysteine Proteases—A Lesson on the Biological Reactivity of Alkynes. Angew. Chem., Int. Ed. 2013, 52 (32), 8210– 8212, DOI: 10.1002/anie.201303544[Crossref], [CAS], Google Scholar110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVeqtb0%253D&md5=18ee2b226627f5bf11f4ac54ff365884Propargyl Amides as Irreversible Inhibitors of Cysteine Proteases-A Lesson on the Biological Reactivity of AlkynesArkona, Christoph; Rademann, JoergAngewandte Chemie, International Edition (2013), 52 (32), 8210-8212CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Ubiquitin like proteins are transferred to target proteins and bound by means of isopeptide bonds in an ATP driven enzymic transfer reaction with thioester intermediates. Modified target proteins are often driven towards degrdn. De-ubiquitinating enzymes (DUB) are capable of hydrolyzing the isopeptide bonds thereby reliberating ubiquitin-like proteins and target proteins. This review discusses the role of DUBs in health and disease.
- 111Sanger, F. The Free Amino Groups of Insulin. Biochem. J. 1945, 39 (5), 507– 515, DOI: 10.1042/bj0390507[Crossref], [PubMed], [CAS], Google Scholar111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaH28XislKmsA%253D%253D&md5=7f87d7cb3a83266868814046972cde6fFree amino groups of insulinSanger, F.Biochemical Journal (1945), 39 (), 507-15CODEN: BIJOAK; ISSN:0264-6021.The free amino groups of proteins and peptides readily form derivs. with 2,4-(O2N)2C6H3F (I) at room temp. Since these derivs. are relatively stable to acid hydrolysis, hydrolysis of the substituted protein enables isolation of 2,4-dinitrophenyl amino acid derivs. which are separable by partition chromatography. This, then, forms the basis of a new method which was applied to the identification and estn. of the free amino groups in insulin. For comparison and control, several derivs. of the naturally occurring amino acids had to be synthesized. To a mixt. of 0.2 g. l-phenylalanine and 0.4 g. NaHCO3 in 5 ml. H2O was added a soln. of 0.4 g. I in 10 ml. EtOH. The mixt. was reacted 2 hrs. at room temp., giving 0.27 g. of N-2,4-dinitrophenyl-l-phenylalanine, C15H13O6N3, m. 186°. α-Acetyl-l-lysine (0.48 g.) and 0.75 g. NaHCO3 in 3 ml. H2O reacted for 4 hrs. at reflux temp. with 0.5 g. 2,4-(O2N)2C6H3Cl (II) in 10 ml. EtOH gave 0.7 g. (75%) of α-acetyl-ε-2,4-dinitrophenyl-l-lysine (III), m. 110°. III refluxed for 3 hrs. with 20% HCl gives ε-2,4-dinitrophenyl-l-lysine-HCl, which seps. from dil. HCl as the monohydrate, C12H16O6N4.HCl.H2O, m. 186°. ε-Benzoyl-l-lysine (1.25 g.) treated with 1.0 g. II gives 2 g. of α-2,4-dinitrophenyl-ε-benzoyl-l-lysine (IV). IV (0.5 g.) hydrolyzed in HOAc and HCl gave 60% of α-2,4-dinitrophenyl-l-lysine, C12H16O6N4, m. 260° (decompn.). l-Tyrosine (V) in the presence of excess I gives a compd. which is presumably O,N-bis(2,4 dinitrophenyl)-l-tyrosine (VI). Reaction of V with II gives, in addn. to VI, a compd. which was probably N-2,4-dinitrophenyl-l-tyrosine. N-Acetyl-l-tyrosine (0.55 g.) treated for 4 hrs. with 2.0 g. II gave 0.1 g. of N-acetyl-O-2,4-dinitrophenyl-l-tyrosine (VII), m. 194°. VII hydrolyzed in 20% HCl gave O-2,4-dinitrophenyl-l-tyrosine, as the hydrate, C15H13O7N3.H2O, m. 202°. On treatment of histidine with excess I only the bis(2,4-dinitrophenyl) deriv. is obtained, indicating that I reacts with the imidazole ring. This is confirmed by the slow reaction of I with α-acetylhistidine and rapid reaction with histidine Me ester to give again a bis deriv. I reacts also with SH groups of S-contg. amino acids but a mixt. of products is apparently obtained. The band rates on the chromatogram for various known 2,4-dinitrophenyl amino acids were detd. for various solvent mixts. and compared with those of the derivs. obtained from hydrolyzed dinitrophenylinsulin. No indicator was necessary since all of the derivs. were colored. Isolation of the bands enabled quant. colorimetric estn. of the derivs., using the known compds. as standards. Application of this technique shows that in an insulin submol. of mol. wt. 12,000, six free amino groups are present; 2 of these are located on glycine residues, 2 on phenylalanine residues, and 2 represent the ε-amino groups of lysine. The results suggest that the insulin submol. is made up of 4 open polypeptide chains, 2 of these having terminal glycyl residues and the other 2 terminal phenylalanyl residues, the chains being bound together most probably by -S-S- linkages.
- 112Terrier, F. Rate and Equilibrium Studies in Jackson-Meisenheimer Complexes. Chem. Rev. 1982, 82 (2), 77– 152, DOI: 10.1021/cr00048a001[ACS Full Text], [CAS], Google Scholar112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XitFKrtrY%253D&md5=47e9957a89dbb9c19be1dbe630cb8315Rate and equilibrium studies in Jackson-Meisenheimer complexesTerrier, FrancoisChemical Reviews (Washington, DC, United States) (1982), 82 (2), 77-152CODEN: CHREAY; ISSN:0009-2665.A review of the title subject and its relation to SNAr reactions with 492 refs.
- 113Terrier, F. Modern Nucleophilic Aromatic Substitution, 1st ed.; Wiley-VCH: Weinheim, 2013.
- 114Kwan, E. E.; Zeng, Y.; Besser, H. A.; Jacobsen, E. N. Concerted Nucleophilic Aromatic Substitutions. Nat. Chem. 2018, 10 (9), 917– 923, DOI: 10.1038/s41557-018-0079-7[Crossref], [PubMed], [CAS], Google Scholar114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlGiurzI&md5=4b75a2efbbd6b64587af174f07ec6d70Concerted nucleophilic aromatic substitutionsKwan, Eugene E.; Zeng, Yuwen; Besser, Harrison A.; Jacobsen, Eric N.Nature Chemistry (2018), 10 (9), 917-923CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Nucleophilic arom. substitution (SNAr) is one of the most widely applied reaction classes in pharmaceutical and chem. research, providing a broadly useful platform for the modification of arom. ring scaffolds. The generally accepted mechanism for SNAr reactions involves a two-step addn.-elimination sequence via a discrete, non-arom. Meisenheimer complex. Here the authors use 12C/13C kinetic isotope effect (KIE) studies and computational analyses to provide evidence that prototypical SNAr reactions in fact proceed through concerted mechanisms. The KIE measurements were made possible by a new technique that leverages the high sensitivity of 19F as an NMR nucleus to quantitate the degree of isotopic fractionation. This sensitive technique permits the measurement of KIEs on 10 mg of natural abundance material in one overnight acquisition. As a result, it provides a practical tool for performing detailed mechanistic analyses of reactions that form or break C-F bonds.
- 115Elbrecht, A.; Chen, Y.; Adams, A.; Berger, J.; Griffin, P.; Klatt, T.; Zhang, B.; Menke, J.; Zhou, G.; Smith, R. G.; Moller, D. E. L-764406 Is a Partial Agonist of Human Peroxisome Proliferator-Activated Receptor γ. The Role of Cys13 in Ligand Binding. J. Biol. Chem. 1999, 274 (12), 7913– 7922, DOI: 10.1074/jbc.274.12.7913[Crossref], [PubMed], [CAS], Google Scholar115https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXitFSkur8%253D&md5=11437562ebe253b2fcf27afbcc7bd597L-764406 is a partial agonist of human peroxisome proliferator-activated receptor γ. The role of Cys313 in ligand bindingElbrecht, Alex; Chen, Yuli; Adams, Alan; Berger, Joel; Griffin, Patrick; Klatt, Tracey; Zhang, Bei; Menke, John; Zhou, Gaochao; Smith, Roy G.; Moller, David E.Journal of Biological Chemistry (1999), 274 (12), 7913-7922CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Insulin-sensitizing thiazolidinedione (TZD) compds. are high affinity ligands for a member of the nuclear receptor family, peroxisome proliferator-activated receptor (PPAR) γ. A scintillation proximity assay for measurement of 3H-radiolabeled TZD binding to human PPARγ under homogeneous conditions was developed. Using this approach, a novel non-TZD compd. (L-764406) was shown to be a potent (apparent binding IC50 of 70 nM) PPARγ ligand. Preincubation of PPARγ with L-764406 prevented binding of the [3H]TZD, suggesting a covalent interaction with the receptor; in addn., structurally related analogs of L-764406, which would be predicted not to interact with PPARγ in a covalent fashion, did not displace [3H]TZD binding to PPARγ. Covalent binding of L-764406 was proven by an obsd. mol. wt. shift of a tryptic PPARγ ligand binding domain (LBD) peptide by mass spectrometric anal. A specific cysteine residue (Cys313 in helix 3 of hPPARγ2) was identified as the attachment site for this compd. In protease protection expts., the liganded receptor adopted a typical agonist conformation. L-764406 exhibited partial agonist activity in cells expressing a chimeric receptor contg. the PPARγ LBD and a cognate reporter gene and also induced the expression of the adipocyte-specific gene aP2 in 3T3-L1 cells. In contrast, L-764406 did not exhibit activity in cells transfected with chimeric receptors contg. PPARα or PPARδ LBDs. The partial agonist properties of L-764406 were also evident in a co-activator assocn. assay, indicating that the increased transcription in cells was co-activator mediated. Thus, L-764406 is a novel non-TZD ligand for PPARγ and is also the first known partial agonist for this receptor. The results suggest a crit. functional role for Cys313, and helix 3, in contributing to ligand binding and subsequent agonist-induced conformational changes.
- 116Leesnitzer, L. M.; Parks, D. J.; Bledsoe, R. K.; Cobb, J. E.; Collins, J. L.; Consler, T. G.; Davis, R. G.; Hull-Ryde, E. A.; Lenhard, J. M.; Patel, L.; Plunket, K. D.; Shenk, J. L.; Stimmel, J. B.; Therapontos, C.; Willson, T. M.; Blanchard, S. G. Functional Consequences of Cysteine Modification in the Ligand Binding Sites of Peroxisome Proliferator Activated Receptors by GW9662. Biochemistry 2002, 41 (21), 6640– 6650, DOI: 10.1021/bi0159581[ACS Full Text], [CAS], Google Scholar116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtF2htLo%253D&md5=4c8b3355a3a3b43577749d7805f85affFunctional Consequences of Cysteine Modification in the Ligand Binding Sites of Peroxisome Proliferator Activated Receptors by GW9662Leesnitzer, Lisa M.; Parks, Derek J.; Bledsoe, Randy K.; Cobb, Jeff E.; Collins, Jon L.; Consler, Thomas G.; Davis, Roderick G.; Hull-Ryde, Emily A.; Lenhard, James M.; Patel, Lisa; Plunket, Kelli D.; Shenk, Jennifer L.; Stimmel, Julie B.; Therapontos, Christina; Willson, Timothy M.; Blanchard, Steven G.Biochemistry (2002), 41 (21), 6640-6650CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-γ (PPARγ), the authors identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC50 vs. PPARγ and was 10- and 600-fold less potent in binding expts. using PPARα and PPARδ, resp. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric anal. of the PPARγ ligand binding domain treated with GW9662 established Cys285 as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARγ. The functional activity of GW9662 as an antagonist of PPARγ was confirmed in an assay of adipocyte differentiation. GW9662 showed essentially no effect on transcription when tested using both full-length PPARδ and PPARα. Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects obsd. in the reporter gene assays. Control activators increased PPAR: RXR heterodimer formation and coactivator binding to both PPARγ and PPARδ. Corepressor binding was decreased. In the case of PPARα, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding. The exptl. data indicate that GW9662 modification of each of the three PPARs results in different functional consequences. The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture expts., suggests that this compd. may be a useful tool for elucidation of the role of PPARγ in biol. processes.
- 117Shearer, B. G.; Wiethe, R. W.; Ashe, A.; Billin, A. N.; Way, J. M.; Stanley, T. B.; Wagner, C. D.; Xu, R. X.; Leesnitzer, L. M.; Merrihew, R. V.; Shearer, T. W.; Jeune, M. R.; Ulrich, J. C.; Willson, T. M. Identification and Characterization of 4-Chloro-N-(2-{[5-Trifluoromethyl)-2-Pyridyl]Sulfonyl}ethyl)Benzamide (GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) Antagonist. J. Med. Chem. 2010, 53 (4), 1857– 1861, DOI: 10.1021/jm900464j[ACS Full Text], [CAS], Google Scholar117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVGisrc%253D&md5=401381858ca225791e8a90c13bb9d5e4Identification and Characterization of 4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide (GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) AntagonistShearer, Barry G.; Wiethe, Robert W.; Ashe, Adam; Billin, Andrew N.; Way, James M.; Stanley, Thomas B.; Wagner, Craig D.; Xu, Robert X.; Leesnitzer, Lisa M.; Merrihew, Raymond V.; Shearer, Todd W.; Jeune, Michael R.; Ulrich, John C.; Willson, Timothy M.Journal of Medicinal Chemistry (2010), 53 (4), 1857-1861CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)GSK3787 (I) was identified as a potent and selective ligand for PPARδ with good pharmacokinetic properties. A detailed binding study using mass spectral anal. confirmed covalent binding to Cys249 within the PPARδ binding pocket. Gene expression studies showed that pyridylsulfone 3 antagonized the transcriptional activity of PPARδ and inhibited basal CPT1a gene transcription. Compd. 3 is a PPARδ antagonist with utility as a tool to elucidate PPARδ cell biol. and pharmacol.
- 118Babaoglu, K.; Simeonov, A.; Irwin, J. J.; Nelson, M. E.; Feng, B.; Thomas, C. J.; Cancian, L.; Costi, M. P.; Maltby, D. A.; Jadhav, A.; Inglese, J.; Austin, C. P.; Shoichet, B. K. Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase. J. Med. Chem. 2008, 51 (8), 2502– 2511, DOI: 10.1021/jm701500e[ACS Full Text], [CAS], Google Scholar118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjtFSksb0%253D&md5=7736429aeafc6b17a5d0182e24df0dd4Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-LactamaseBabaoglu, Kerim; Simeonov, Anton; Irwin, John J.; Nelson, Michael E.; Feng, Brian; Thomas, Craig J.; Cancian, Laura; Costi, M. Paola; Maltby, David A.; Jadhav, Ajit; Inglese, James; Austin, Christopher P.; Shoichet, Brian K.Journal of Medicinal Chemistry (2008), 51 (8), 2502-2511CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)High-throughput screening (HTS) is widely used in drug discovery. Esp. for screens of unbiased libraries, false positives can dominate "hit lists"; their origins are much debated. Here we det. the mechanism of every active hit from a screen of 70,563 unbiased mols. against β-lactamase using quant. HTS (qHTS). Of the 1274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting β-lactams. Mass spectra, counter-screens, and crystallog. identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to mol. docking to prioritize mols. from the same library for testing at higher concns. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 μM. These results suggest that it may be the phys. behavior of org. mols., not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens.
- 119Patterson, J. T.; Asano, S.; Li, X.; Rader, C.; Barbas, C. F. Improving the Serum Stability of Site-Specific Antibody Conjugates with Sulfone Linkers. Bioconjugate Chem. 2014, 25 (8), 1402– 1407, DOI: 10.1021/bc500276m[ACS Full Text], [CAS], Google Scholar119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht12gu77I&md5=1692372289fc0c7dabf1374851b5ccd6Improving the Serum Stability of Site-Specific Antibody Conjugates with Sulfone LinkersPatterson, James T.; Asano, Shigehiro; Li, Xiuling; Rader, Christoph; Barbas, Carlos F., IIIBioconjugate Chemistry (2014), 25 (8), 1402-1407CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Current routes for synthesizing antibody-drug conjugates commonly rely on maleimide linkers to react with cysteine thiols. However, thioether exchange with metabolites and serum proteins can compromise conjugate stability and diminish in vivo efficacy. We report the application of a phenyloxadiazole sulfone linker for the prepn. of trastuzumab conjugates. This sulfone linker site-specifically labeled engineered cysteine residues in THIOMABs and improved antibody conjugate stability in human plasma at sites previously shown to be labile for maleimide conjugates. Similarly, sulfone conjugation with selenocysteine in an anti-ROR1 scFv-Fc improved human plasma stability relative to maleimide conjugation. Kinetically controlled labeling of a THIOMAB contg. two cysteine substitutions was also achieved, offering a strategy for producing antibody conjugates with expanded valency.
- 120Zhang, D.; Devarie-Baez, N. O.; Li, Q.; Lancaster, J. R.; Xian, M. Methylsulfonyl Benzothiazole (MSBT): A Selective Protein Thiol Blocking Reagent. Org. Lett. 2012, 14 (13), 3396– 3399, DOI: 10.1021/ol301370s[ACS Full Text], [CAS], Google Scholar120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xot1antL8%253D&md5=6e785b51e6288197559db5a91a982a9aMethylsulfonyl Benzothiazole (MSBT): A Selective Protein Thiol Blocking ReagentZhang, Dehui; Devarie-Baez, Nelmi O.; Li, Qian; Lancaster, Jack R.; Xian, MingOrganic Letters (2012), 14 (13), 3396-3399CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)A new thiol blocking reagent, methylsulfonyl benzothiazole, was discovered. This reagent showed good selectivity and high reactivity for protein thiols.
- 121Toda, N.; Asano, S.; Barbas, C. F. Rapid, Stable, Chemoselective Labeling of Thiols with Julia–Kocieński-like Reagents: A Serum-Stable Alternative to Maleimide-Based Protein Conjugation. Angew. Chem., Int. Ed. 2013, 52 (48), 12592– 12596, DOI: 10.1002/anie.201306241[Crossref], [CAS], Google Scholar121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1Wms7%252FF&md5=ef91e1e933d116482b9e886c94c6cdd0Rapid, stable, chemoselective labeling of thiols with Julia-Kocienski-like reagents: A serum-stable alternative to maleimide-based protein conjugationToda, Narihiro; Asano, Shigehiro; Barbas, Carlos F., IIIAngewandte Chemie, International Edition (2013), 52 (48), 12592-12596CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)We have developed a class of sulfone derivs. for applications in protein conjugation chem., and we have compared the newly synthesized conjugates to maleimide conjugates. Methylsulfonyl-functionalized five-membered cyclic compds., such as phenyltetrazole or phenyloxadiazole, reacted rapidly and specifically with thiols in small mols. and proteins with exquisite chemoselectivity at biol. relevant pH values (pH 5.8 - 8.0). Designer heteroarom. sulfones allowed for the selective introduction of a fluorophore and poly(ethylene) glycol chains (PEGylation), and provided protein conjugates with superior stability compared to maleimide-conjugated proteins in human plasma. Given the speed, selectivity, and stability of the sulfone-cysteine reactions described herein, we anticipate that this "thiol-Click" approach will find broad application in peptide and protein chem. and for the development of antibody drug conjugates.
- 122Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y.-S.; Pentelute, B. L. A Perfluoroaryl-Cysteine SNAr Chemistry Approach to Unprotected Peptide Stapling. J. Am. Chem. Soc. 2013, 135 (16), 5946– 5949, DOI: 10.1021/ja400119t[ACS Full Text], [CAS], Google Scholar122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlsV2gtrc%253D&md5=126ac191059b0703757ef7838d90e183A Perfluoroaryl-Cysteine SNAr Chemistry Approach to Unprotected Peptide StaplingSpokoyny, Alexander M.; Zou, Yekui; Ling, Jingjing J.; Yu, Hongtao; Lin, Yu-Shan; Pentelute, Bradley L.Journal of the American Chemical Society (2013), 135 (16), 5946-5949CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors report the discovery of a facile transformation between perfluoroarom. mols. and a cysteine thiolate, which is arylated at room temp. This new approach enabled the authors to selectively modify cysteine residues in unprotected peptides, providing access to variants contg. rigid perfluoroarom. staples. This stapling modification performed on a peptide sequence designed to bind the C-terminal domain of an HIV-1 (C-CA) showed enhancement in binding, cell permeability, and proteolytic stability properties, as compared to the unstapled analog. Importantly, chem. stability of the formed staples allowed the authors to use this motif in the native chem. ligation-mediated synthesis of a small protein affibody that is capable of binding the human epidermal growth factor 2 receptor.
- 123Alapour, S.; de la Torre, B. G.; Ramjugernath, D.; Koorbanally, N. A.; Albericio, F. Application of Decafluorobiphenyl (DFBP) Moiety as a Linker in Bioconjugation. Bioconjugate Chem. 2018, 29 (2), 225– 233, DOI: 10.1021/acs.bioconjchem.7b00800[ACS Full Text], [CAS], Google Scholar123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFKjur0%253D&md5=80ad66589d3d56dd49d8b16a1a8de884Application of Decafluorobiphenyl (DFBP) Moiety as a Linker in BioconjugationAlapour, Saba; de la Torre, Beatriz G.; Ramjugernath, Deresh; Koorbanally, Neil A.; Albericio, FernandoBioconjugate Chemistry (2018), 29 (2), 225-233CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)A review. Considerable attention has been devoted to fluorinated compds. due to their unique and interesting properties. Many modern pharmaceuticals contain fluorinated substituents, which are commonly synthesized using selective fluorinating reagents. Decafluorobiphenyl (DFBP) as a fluorinated linker is susceptible to nucleophilic attack. This nucleophilic reaction has been widely studied using various nucleophiles. Sulfur and nitrogen contg. nucleophiles have been of particular interest, esp. in bioconjugated reactions. This review focuses on the SNAr reactivity of DFBP in formation of C-X (X = S, N) bonds, to be applied in bioconjugation in org. chem. The review aims to highlight the crucial factors that govern the chem. behind the activation of F-CAr-CAr-F bonds as a linker in the synthesis of novel peptides, proteins, and biologics.
- 124Brown, S. P.; Smith, A. B. Peptide/Protein Stapling and Unstapling: Introduction of s-Tetrazine, Photochemical Release, and Regeneration of the Peptide/Protein. J. Am. Chem. Soc. 2015, 137 (12), 4034– 4037, DOI: 10.1021/ja512880g[ACS Full Text], [CAS], Google Scholar124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvFWjt7w%253D&md5=55fd8c77ca0926af46e6f9a341a45008Peptide/Protein Stapling and Unstapling: Introduction of s-Tetrazine, Photochemical Release, and Regeneration of the Peptide/ProteinBrown, Stephen P.; Smith, Amos B., IIIJournal of the American Chemical Society (2015), 137 (12), 4034-4037CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protocols have been achieved that permit facile introduction of s-tetrazine into unprotected peptides and the protein, thioredoxin, between two cysteine sulfhydryl groups (i.e., staple), followed by photochem. release (i.e., unstaple) and regeneration of the peptide/ protein upon removal of the cyano groups from the derived bisthiocyanate. The S,S-tetrazine macrocycles in turn provide a convenient handle for probe introduction by exploiting the inverse electron demand Diels-Alder reactivity of the tetrazine.
- 125Roberts, D. W.; Aptula, A. O. Electrophilic Reactivity and Skin Sensitization Potency of SNAr Electrophiles. Chem. Res. Toxicol. 2014, 27 (2), 240– 246, DOI: 10.1021/tx400355n[ACS Full Text], [CAS], Google Scholar125https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjtVWmsA%253D%253D&md5=30a013cec875f8fa3d482421dada4e88Electrophilic Reactivity and Skin Sensitization Potency of SNAr ElectrophilesRoberts, D. W.; Aptula, A. O.Chemical Research in Toxicology (2014), 27 (2), 240-246CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)The authors published in 2011 a quant. mechanistic model (QMM) for skin sensitization potency of SNAr electrophiles in the mouse local lymph node assay (LLNA). In this model, potency was correlated with a combination of σ* for the leaving group and the total σ- values of the other substituents in the arom. ring. Shortly afterward Natsch et al. published a kinetic study in which rate consts. were detd. for reactions of SNAr electrophiles with the cysteine-based peptide Ac-RFAACAA (Cys-peptide) that is used in the direct peptide reactivity assay (DPRA), and correlations were sought between these rate consts. and sensitization potency in the LLNA. These two publications together have enabled the present study, aiming to develop a linear free energy relationship (LFER) correlating Cys-peptide reactivity with a reactivity parameter (RP) based on a combination of σ* and σ- substituent consts. and, by analyzing differences between the QMM based on RP and the QMM based on Cys-peptide rate consts., to gain further insights into the underlying chem. of skin sensitization. For the 2,4-dinitro-X-substituted benzenes (DNXB), the rate consts. of Natsch et al. are well correlated with the reactivity parameter used in the authors' earlier work, with two outliers. These are the compds. with X = F and X = SCN, which are both substantially more reactive toward Cys-peptide than predicted from comparison of their RP values with those of the other DNXB compds. These two compds. are both neg. outliers from a correlation of sensitization potency with exptl. rate consts., but fit well to the correlation of sensitization potency with RP values. With these two compds. excluded, sensitization potency is well correlated with the exptl. rate consts. for the DNXB compds. (X = SO3-, I, Br, Cl) together with 2,4-dichloro-1-nitrobenzene and 1,3,4,5-tetrachloro-2,6-dicyanobenzene. The regression equation is pEC3 = 0.88 log k + 4.03, R2 = 0.966. The implication of DNFB being an outlier is that the model Cys-peptide nucleophile is substantially more sterically hindered than the cutaneous nucleophile(s) involved in the sensitization process. The pattern seen with 2,4-dinitrothiocyanatobenzene suggests that this compd. reacts as an SNAr electrophile in the sensitization process, but by a different pathway, acting as a CN transfer agent, with the model Cys-peptide. For two further compds., 2,4,6-trinitrochlorobenzene and 2,4,6-trinitrobenzenesulfonate, the Cys-peptide rate consts. are well predicted by the reactivity parameter based on displacement of the Cl or SO3- substituent, but their sensitization potency is underestimated by both the Cys-peptide rate const. and this reactivity parameter. However, potency of these two compds. is well predicted by a reactivity parameter calcd. on the basis of displacement of the 2-nitro group. This is interpreted as a case of sensitization being driven by the thermodynamically favored rather than the kinetically favored reaction product.
- 126Hwang, J. Y.; Huang, W.; Arnold, L. A.; Huang, R.; Attia, R. R.; Connelly, M.; Wichterman, J.; Zhu, F.; Augustinaite, I.; Austin, C. P.; Inglese, J.; Johnson, R. L.; Guy, R. K. Methylsulfonylnitrobenzoates, a New Class of Irreversible Inhibitors of the Interaction of the Thyroid Hormone Receptor and Its Obligate Coactivators That Functionally Antagonizes Thyroid Hormone. J. Biol. Chem. 2011, 286 (14), 11895– 11908, DOI: 10.1074/jbc.M110.200436[Crossref], [PubMed], [CAS], Google Scholar126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktVKqtro%253D&md5=a968a083f7c5718c2e03d5ef0a3b25aeMethylsulfonylnitrobenzoates, a New Class of Irreversible Inhibitors of the Interaction of the Thyroid Hormone Receptor and its Obligate Coactivators that Functionally Antagonizes Thyroid HormoneHwang, Jong-Yeon; Huang, Wen-Wei; Arnold, Leggy A.; Huang, Rui-Li; Attia, Ramy R.; Connelly, Michele; Wichterman, Jennifer; Zhu, Fang-Yi; Augustinaite, Indre; Austin, Christopher P.; Inglese, James; Johnson, Ronald L.; Guy, R. KiplinJournal of Biological Chemistry (2011), 286 (14), 11895-11908CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metab. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a no. of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the assocn. of TRβ with steroid receptor coactivator 2 (SRC2), the authors identified a novel methylsulfonylnitrobenzoate (MSNB)-contg. series that blocks this interaction at micromolar concns. Here the authors have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRβ antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum anal. suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRβ to disrupt SRC2 assocn.
- 127Arnold, L. A.; Kosinski, A.; Estébanez-Perpiñá, E.; Guy, R. K. Inhibitors of the Interaction of a Thyroid Hormone Receptor and Coactivators: Preliminary Structure–Activity Relationships. J. Med. Chem. 2007, 50 (22), 5269– 5280, DOI: 10.1021/jm070556y[ACS Full Text], [CAS], Google Scholar127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFWktr%252FE&md5=396a0fbc7879935595d695c4e6c56bbaInhibitors of the Interaction of a Thyroid Hormone Receptor and Coactivators: Preliminary Structure-Activity RelationshipsArnold, Leggy A.; Kosinski, Aaron; Estebanez-Perpina, Eva; Fletterick, Robert J.; Guy, R. KiplinJournal of Medicinal Chemistry (2007), 50 (22), 5269-5280CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The modulation of gene regulation by blocking the interaction between the thyroid receptor (TR) and obligate coregulators has been reported recently with the discovery of the lead compd. 3-(dimethylamino)-1-(4-hexylphenyl)propan-1-one. Herein the authors report studies aimed at optimization of this initial hit to det. the basic parameters of the structure-activity relationships and clarify the mechanism of action. These studies provided new insights, showing that activity and TRβ isoform selectivity is highly correlated with the structural compn. of these covalent inhibitors.
- 128Visperas, P. R.; Winger, J. A.; Horton, T. M.; Shah, N. H.; Aum, D. J.; Tao, A.; Barros, T.; Yan, Q.; Wilson, C. G.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Modification by Covalent Reaction or Oxidation of Cysteine Residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding. Biochem. J. 2015, 465 (1), 149– 161, DOI: 10.1042/BJ20140793[Crossref], [PubMed], [CAS], Google Scholar128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFWiu7vE&md5=219ac232370be3247dee4e4bb7582925Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide bindingVisperas, Patrick R.; Winger, Jonathan A.; Horton, Timothy M.; Shah, Neel H.; Aum, Diane J.; Tao, Alyssa; Barros, Tiago; Yan, Qingrong; Wilson, Christopher G.; Arkin, Michelle R.; Weiss, Arthur; Kuriyan, JohnBiochemical Journal (2015), 465 (1), 149-161CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Zeta-chain assocd. protein of 70 kDa (ZAP-70) and spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signalling resp. They are recruited, via their tandem-SH2 (Src-homol. domain 2) domains, to doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their crit. roles in immune signalling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small mols. can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys39 in ZAP-70, Cys206 in Syk) that is necessary for inhibition by two of these compds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of H2O2 and these two cysteine residues are also necessary for inhibition by H2O2. Our findings suggest a mechanism by which the reactive oxygen species generated during responses to antigen could attenuate signalling through these kinases and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains.
- 129Visperas, P. R.; Wilson, C. G.; Winger, J. A.; Yan, Q.; Lin, K.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput Screen. SLAS Discov. 2017, 22 (3), 324– 331, DOI: 10.1177/1087057116681407[Crossref], [PubMed], [CAS], Google Scholar129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFKksLc%253D&md5=49eacea4b3bbba376aa7fd7785ef0e22Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput ScreenVisperas, Patrick R.; Wilson, Christopher G.; Winger, Jonathan A.; Yan, Qingrong; Lin, Kevin; Arkin, Michelle R.; Weiss, Arthur; Kuriyan, JohnSLAS Discovery (2017), 22 (3), 324-331CODEN: SDLIBT; ISSN:2472-5560. (Sage Publications)ZAP-70 is a crit. mol. in the transduction of T cell antigen receptor signaling and the activation of T cells. Upon activation of the T cell antigen receptor, ZAP-70 is recruited to the intracellular ζ-chains of the T cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T cell antigen receptor. The screen is based on a fluorescence polarization assay for peptide binding to ZAP-70.
- 130Shannon, D. A.; Banerjee, R.; Webster, E. R.; Bak, D. W.; Wang, C.; Weerapana, E. Investigating the Proteome Reactivity and Selectivity of Aryl Halides. J. Am. Chem. Soc. 2014, 136 (9), 3330– 3333, DOI: 10.1021/ja4116204[ACS Full Text], [CAS], Google Scholar130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXislentLo%253D&md5=b8859e8d24d051fb62ab4dfba7431fe1Investigating the Proteome Reactivity and Selectivity of Aryl HalidesShannon, D. Alexander; Banerjee, Ranjan; Webster, Elizabeth R.; Bak, Daniel W.; Wang, Chu; Weerapana, EranthieJournal of the American Chemical Society (2014), 136 (9), 3330-3333CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein-reactive electrophiles are crit. to chem. proteomic applications including activity-based protein profiling, site-selective protein modification, and covalent inhibitor development. Here, we explore the protein reactivity of a panel of aryl halides that function through a nucleophilic arom. substitution (SNAr) mechanism. We show that the reactivity of these electrophiles can be finely tuned by varying the substituents on the aryl ring. We identify p-chloro- and fluoronitrobenzenes and dichlorotriazines as covalent protein modifiers at low micromolar concns. Interestingly, investigating the site of labeling of these electrophiles within complex proteomes identified p-chloronitrobenzene as highly cysteine selective, whereas the dichlorotriazine favored reactivity with lysines. These studies illustrate the diverse reactivity and amino-acid selectivity of aryl halides and enable the future application of this class of electrophiles in chem. proteomics.
- 131Schardon, C. L.; Tuley, A.; Er, J. A. V.; Swartzel, J. C.; Fast, W. Selective Covalent Protein Modification by 4-Halopyridines through Catalysis. ChemBioChem 2017, 18 (15), 1551– 1556, DOI: 10.1002/cbic.201700104[Crossref], [PubMed], [CAS], Google Scholar131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKhsbrE&md5=c71e0d7b061645a374919194678cab1aSelective Covalent Protein Modification by 4-Halopyridines through CatalysisSchardon, Christopher L.; Tuley, Alfred; Er, Joyce A. V.; Swartzel, Jake C.; Fast, WalterChemBioChem (2017), 18 (15), 1551-1556CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)We have investigated 4-halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chem. probes. Nonenzymic reactivity of 4-chloropyridine with amino acids and thiols was ranked with respect to common covalent protein-modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the pos. charged pyridinium. Diverse, fragment-sized 4-halopyridines inactivated human dimethylarginine dimethylaminohydrolase-1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off-pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2-fluoromethyl-substituted 4-chloropyridines demonstrated that the pKa and kinact/KI values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4-Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.
- 132Fairhurst, R. A.; Knoepfel, T.; Leblanc, C.; Buschmann, N.; Gaul, C.; Blank, J.; Galuba, I.; Trappe, J.; Zou, C.; Voshol, J.; Genick, C.; Brunet-Lefeuvre, P.; Bitsch, F.; Graus-Porta, D.; Furet, P. Approaches to Selective Fibroblast Growth Factor Receptor 4 Inhibition through Targeting the ATP-Pocket Middle-Hinge Region. MedChemComm 2017, 8, 1604– 1613, DOI: 10.1039/C7MD00213K[Crossref], [PubMed], [CAS], Google Scholar132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXps1GlsL8%253D&md5=04f1aff88385b915e2672509dd3f6954Approaches to selective fibroblast growth factor receptor 4 inhibition through targeting the ATP-pocket middle-hinge regionFairhurst, Robin A.; Knoepfel, Thomas; Leblanc, Catherine; Buschmann, Nicole; Gaul, Christoph; Blank, Jutta; Galuba, Inga; Trappe, Jorg; Zou, Chao; Voshol, Johannes; Genick, Christine; Brunet-Lefeuvre, Peggy; Bitsch, Francis; Graus-Porta, Diana; Furet, PascalMedChemComm (2017), 8 (8), 1604-1613CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)A diverse range of selective FGFR4 inhibitor hit series were identified using unbiased screening approaches and by the modification of known kinase inhibitor scaffolds. In each case the origin of the selectivity was consistent with an interaction with a poorly conserved cysteine residue within the middle-hinge region of the kinase domain of FGFR4, at position 552. Targeting this region identified a non-covalent diaminopyrimidine series differentiating by size, an irreversible-covalent inhibitor in which Cys552 undergoes an SNAr reaction with a 2-chloropyridine, and a reversible-covalent inhibitor series in which Cys552 forms a hemithioacetal adduct with a 2-formyl naphthalene. In addn., the introduction of an acrylamide into a known FGFR scaffold identified a pan-FGFR inhibitor which reacted with both Cys552 and a second poorly conserved cysteine on the P-loop of FGFR4 at position 477 which is present in all four FGFR family members.
- 133Hou, W.; Ren, Y.; Zhang, Z.; Sun, H.; Ma, Y.; Yan, B. Novel Quinazoline Derivatives Bearing Various 6-Benzamide Moieties as Highly Selective and Potent EGFR Inhibitors. Bioorg. Med. Chem. 2018, 26 (8), 1740– 1750, DOI: 10.1016/j.bmc.2018.02.022[Crossref], [PubMed], [CAS], Google Scholar133https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjvFyntLw%253D&md5=a203ff10067b1b141637f110f0781695Novel quinazoline derivatives bearing various 6-benzamide moieties as highly selective and potent EGFR inhibitorsHou, Weijie; Ren, Yan; Zhang, Zhenhua; Sun, Huan; Ma, Yongfen; Yan, BoBioorganic & Medicinal Chemistry (2018), 26 (8), 1740-1750CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)A series of novel quinazoline derivs. bearing various C-6 benzamide substituents were synthesized and evaluated as EGFR inhibitors, and most showed significant inhibitory potency against EGFR kinase. In particular, compd. 6g possessed potent inhibitory activity against EGFR wild-type (IC50 = 5 nM), and strong antiproliferative activity against HCC827 and Ba/F3 (L858R) cell lines. Kinase profiling against a panel of 365 kinases showed that 6g was highly selective for EGFR. Furthermore, 6g showed desirable properties in assays of liver microsome metabolic stability and cytochromes P 450 inhibition and preliminary pharmacokinetic study. The overall attractive profile of 6g made it an interesting compd. for further development.
- 134Juchum, M.; Günther, M.; Laufer, S. A. Fighting Cancer Drug Resistance: Opportunities and Challenges for Mutation-Specific EGFR Inhibitors. Drug Resist. Updates 2015, 20, 12– 28, DOI: 10.1016/j.drup.2015.05.002[Crossref], [PubMed], [CAS], Google Scholar134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2Mfot1agsA%253D%253D&md5=e0f18db0613cbf9ddee3021ba7e1bc80Fighting cancer drug resistance: Opportunities and challenges for mutation-specific EGFR inhibitorsJuchum Michael; Gunther Marcel; Laufer Stefan ADrug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy (2015), 20 (), 12-28 ISSN:.Multiple mutations in the EGFR gene are a major cause for the failure of Erlotinib and Gefitinib in the treatment of patients harboring non-small-cell lung cancer (NSCLC) who initially responded to this therapy. The development of these tyrosine kinase inhibitors (TKIs) is going back to the early 90s, where cancer was widely considered and fully treated as a disease of an organ. Fundamental gain of knowledge in cell biology in general and cancer genetics in particular led us to where we currently stand: cancer is a disease that originates in the genome. Fast and affordable gene sequencing paved the way and opened our eyes for the genetic instability of many cancers, particularly EGFR driven NSCLC. This might allow highly rational and personal therapies by aiming at a very particular wild type and mutant kinase pattern. However, the paradigm "one disease - one target - one drug" is currently challenged. Both activating and deactivating EGFR mutations are known to render the development of novel targeted drugs difficult. Among all lung adenocarcinomas, only 20% are driven by EGFR and only a subpopulation has an activating mutation (e.g. L858R), making them sensitive to first generation EGFR inhibitors. Unfortunately, most of them acquire second deactivating mutations (e.g. T790M) during treatment, leading to a complete loss of response. Are specific inhibitors of the double EGFR mutant L858R/T790M the magic bullet? Much scientific evidence but also high expectations justify this approach. Structural biology of EGFR mutants constitutes the basis for highly rational approaches. Second generation pan EGFR inhibitors inhibiting wild type (WT) and mutant EGFR like Afatinib suffer from dose-limiting adverse effects. Inhibition of WT EGFR is considered to be the culprit. Third generation EGFR inhibitors follow two strategies. Mutant selectivity and improved target residential time. These inhibitors display high mutant selectivity and irreversible binding patterns while sparing WT EGFR activity, hence enhancing tumor selectivity while minimizing adverse effects. Third generation EGFR inhibitors are still undergoing preclinical and clinical evaluation. The most advanced are Rociletinib and AZD9291 which displayed encouraging preliminary clinical phase II data regarding response and adverse effects. In the current review we show both a medicinal chemists' approach toward the design of third generation EGFR inhibitors as well as a detailed overview of the development of EGFR inhibitors over the last decade. High interdisciplinary approaches, such as structural biology and time-resolved tumor genetics pave the way toward the development of drugs that target EGFR mutants. This might lead to highly effective targeted and personalized therapies with enhanced response rates for a minor cohort of patients which have to undergo continuous gene sequencing, hence enabling therapies with tailor-made TKIs.
- 135Chen, K. X.; Lesburg, C. A.; Vibulbhan, B.; Yang, W.; Chan, T.-Y.; Venkatraman, S.; Velazquez, F.; Zeng, Q.; Bennett, F.; Anilkumar, G. N.; Duca, J.; Jiang, Y.; Pinto, P.; Wang, L.; Huang, Y.; Selyutin, O.; Gavalas, S.; Pu, H.; Agrawal, S.; Feld, B.; Huang, H.-C.; Li, C.; Cheng, K.-C.; Shih, N.-Y.; Kozlowski, J. A.; Rosenblum, S. B.; Njoroge, F. G. A Novel Class of Highly Potent Irreversible Hepatitis C Virus NS5B Polymerase Inhibitors. J. Med. Chem. 2012, 55 (5), 2089– 2101, DOI: 10.1021/jm201322r[ACS Full Text], [CAS], Google Scholar135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntFGlsg%253D%253D&md5=2da02f6d3c59719f0a1b7646f1ef60cfA Novel Class of Highly Potent Irreversible Hepatitis C Virus NS5B Polymerase InhibitorsChen, Kevin X.; Lesburg, Charles A.; Vibulbhan, Bancha; Yang, Weiying; Chan, Tin-Yau; Venkatraman, Srikanth; Velazquez, Francisco; Zeng, Qingbei; Bennett, Frank; Anilkumar, Gopinadhan N.; Duca, Jose; Jiang, Yueheng; Pinto, Patrick; Wang, Li; Huang, Yuhua; Selyutin, Oleg; Gavalas, Stephen; Pu, Haiyan; Agrawal, Sony; Feld, Boris; Huang, Hsueh-Cheng; Li, Cheng; Cheng, Kuo-Chi; Shih, Neng-Yang; Kozlowski, Joseph A.; Rosenblum, Stuart B.; Njoroge, F. GeorgeJournal of Medicinal Chemistry (2012), 55 (5), 2089-2101CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Starting from indole-based C-3 pyridone HCV NS5B polymerase inhibitor I, structure-activity relationship (SAR) investigations of the indole N-1 benzyl moiety were performed. This study led to the discovery of irreversible inhibitors with p-fluoro-sulfone- or p-fluoro-nitro-substituted N-1 benzyl groups which achieved breakthrough replicon assay potency (EC50 = 1 nM). The formation of a covalent bond with adjacent cysteine-366 thiol was was proved by mass spectroscopy and X-ray crystal structure studies. The C-5 Et C-2 carboxylic acid deriv. II had an excellent oral area-under-the-curve (AUC) of 18 μM·h (10 mg/kg). Its oral exposure in monkeys and dogs was also very good. The NMR ALARM assay, mass spectroscopy expts., in vitro counter screening, and toxicol. assays demonstrated that the covalent bond formation between compd. II and the protein was highly selective and specific. The overall excellent profile of II made it an interesting candidate for further investigation.
- 136Powers, J. P.; Piper, D. E.; Li, Y.; Mayorga, V.; Anzola, J.; Chen, J. M.; Jaen, J. C.; Lee, G.; Liu, J.; Peterson, M. G.; Tonn, G. R.; Ye, Q.; Walker, N. P. C.; Wang, Z. SAR and Mode of Action of Novel Non-Nucleoside Inhibitors of Hepatitis C NS5b RNA Polymerase. J. Med. Chem. 2006, 49 (3), 1034– 1046, DOI: 10.1021/jm050859x[ACS Full Text], [CAS], Google Scholar136https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XksV2ltA%253D%253D&md5=722ac0edb5dabb720e30475ef5925d5fSAR and Mode of Action of Novel Non-Nucleoside Inhibitors of Hepatitis C NS5b RNA PolymerasePowers, Jay P.; Piper, Derek E.; Li, Yang; Mayorga, Veronica; Anzola, John; Chen, James M.; Jaen, Juan C.; Lee, Gary; Liu, Jinqian; Peterson, M. Greg; Tonn, George R.; Ye, Qiuping; Walker, Nigel P. C.; Wang, ZhulunJournal of Medicinal Chemistry (2006), 49 (3), 1034-1046CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochem. potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases. The structures of the complexes formed between several of these inhibitors and HCV NS5b were detd. by x-ray crystallog., and the inhibitors were found to bind in an allosteric binding site sep. from the active site. Structure-activity relationships and structural studies have identified the mechanism of action for compds. in this series, several of which possess drug-like properties, as unique, reversible, covalent inhibitors of HCV NS5b.
- 137Huth, J. R.; Mendoza, R.; Olejniczak, E. T.; Johnson, R. W.; Cothron, D. A.; Liu, Y.; Lerner, C. G.; Chen, J.; Hajduk, P. J. ALARM NMR: A Rapid and Robust Experimental Method to Detect Reactive False Positives in Biochemical Screens. J. Am. Chem. Soc. 2005, 127 (1), 217– 224, DOI: 10.1021/ja0455547[ACS Full Text], [CAS], Google Scholar137https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVCrurrF&md5=466332d4bdcddf6bc8a630aacbbb32efALARM NMR: a rapid and robust experimental method to detect reactive false positives in biochemical screensHuth, Jeffrey R.; Mendoza, Renaldo; Olejniczak, Edward T.; Johnson, Robert W.; Cothron, Darlene A.; Liu, Yaya; Lerner, Claude G.; Chen, Jun; Hajduk, Philip J.Journal of the American Chemical Society (2005), 127 (1), 217-224CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)High-throughput screening (HTS) of large compd. collections typically results in numerous small mol. hits that must be carefully evaluated to identify valid drug leads. Although several filtering mechanisms and other tools exist that can assist the chemist in this process, it is often the case that costly synthetic resources are expended in pursuing false positives. We report here a rapid and reliable NMR-based method for identifying reactive false positives including those that oxidize or alkylate a protein target. Importantly, the reactive species need not be the parent compd., as both reactive impurities and breakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactive mols. by NMR) and is based on monitoring DTT-dependent 13C chem. shift changes of the human La antigen in the presence of a test compd. or mixt. Extensive validation has been performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity. This included comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing a collection of more than 3500 compds. contg. HTS hits from 23 drug targets. The data show that current in silico filtering tools fail to identify more than half of the compds. that can act via reactive mechanisms. Significantly, we show how ALARM NMR data has been crit. in identifying reactive compds. that would otherwise have been prioritized for lead optimization. In addn., a new filtering tool has been developed on the basis of the ALARM NMR data that can augment current in silico programs for identifying nuisance compds. and improving the process of hit triage.
- 138Boelsterli, U. A.; Ho, H. K.; Zhou, S.; Leow, K. Y. Bioactivation and Hepatotoxicity of Nitroaromatic Drugs. Curr. Drug Metab. 2006, 7, 715– 727, DOI: 10.2174/138920006778520606[Crossref], [PubMed], [CAS], Google Scholar138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVWgt7rF&md5=21e155ba17d1ad097e7652a5d22728b3Bioactivation and hepatotoxicity of nitroaromatic drugsBoelsterli, Urs A.; Ho, Han Kiat; Zhou, Shufeng; Leow, Koon YeowCurrent Drug Metabolism (2006), 7 (7), 715-727CODEN: CDMUBU; ISSN:1389-2002. (Bentham Science Publishers Ltd.)A review. Certain drugs contg. a nitroarom. moiety (e.g., tolcapone, nimesulide, nilutamide, flutamide, nitrofurantoin) have been assocd. with organ-selective toxicity including rare cases of idiosyncratic liver injury. What they have in common is the potential for multistep nitroreductive bioactivation (6-electron transfer) that produces the potentially hazardous nitroanion radical, nitroso intermediate, and N-hydroxy deriv. These intermediates have been assocd. with increased oxidant stress and targeting of nucleophilic residues on proteins and nucleic acids. However, other mechanisms including the formation of oxidative metabolites and mitochondrial liability, as well as inherent toxicokinetic properties, also det. the drugs' overall potency. Therefore, structural modification not only of the nitro moiety but also of ring substituents can greatly reduced toxicity. Novel concepts have revealed that, besides the classical microsomal nitroreductases, cytosolic and mitochondrial enzymes including nitric oxide synthase can also bioactivate certain nitroarenes (nilutamide). Furthermore, animal models of silent mitochondrial dysfunction have demonstrated that a mitochondrial oxidant stress posed by certain nitroarom. drugs (nimesulide) can produce significant mitochondrial injury if superimposed on a genetic mitochondrial abnormality. Finally, there may be mechanisms for all nitroarom. drugs that do not involve bioactivation of the nitro group, e.g., AHR interactions with flutamide. Taken together, the focus of research on the hepatic toxicity of nitroarene-contg. drugs has shifted over the past years from the identification of the reactive intermediates generated during the bioreductive pathway to the underlying biomechanisms of liver injury. Most likely one of the next paradigm shifts will include the identification of determinants of susceptibility to nitroarom. drug-induced hepatotoxicity.
- 139Zeng, Q.; Nair, A. G.; Rosenblum, S. B.; Huang, H.-C.; Lesburg, C. A.; Jiang, Y.; Selyutin, O.; Chan, T.-Y.; Bennett, F.; Chen, K. X.; Venkatraman, S.; Sannigrahi, M.; Velazquez, F.; Duca, J. S.; Gavalas, S.; Huang, Y.; Pu, H.; Wang, L.; Pinto, P.; Vibulbhan, B.; Agrawal, S.; Ferrari, E.; Jiang, C.; Li, C.; Hesk, D.; Gesell, J.; Sorota, S.; Shih, N.-Y.; Njoroge, F. G.; Kozlowski, J. A. Discovery of an Irreversible HCV NS5B Polymerase Inhibitor. Bioorg. Med. Chem. Lett. 2013, 23 (24), 6585– 6587, DOI: 10.1016/j.bmcl.2013.10.060[Crossref], [PubMed], [CAS], Google Scholar139https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVWns7zK&md5=15a6315c05dd2c4c1c55420e317756ecDiscovery of an irreversible HCV NS5B polymerase inhibitorZeng, Qingbei; Nair, Anilkumar G.; Rosenblum, Stuart B.; Huang, Hsueh-Cheng; Lesburg, Charles A.; Jiang, Yueheng; Selyutin, Oleg; Chan, Tin-Yau; Bennett, Frank; Chen, Kevin X.; Venkatraman, Srikanth; Sannigrahi, Mousumi; Velazquez, Francisco; Duca, Jose S.; Gavalas, Stephen; Huang, Yuhua; Pu, Haiyan; Wang, Li; Pinto, Patrick; Vibulbhan, Bancha; Agrawal, Sony; Ferrari, Eric; Jiang, Chuan-kui; Li, Cheng; Hesk, David; Gesell, Jennifer; Sorota, Steve; Shih, Neng-Yang; Njoroge, F. George; Kozlowski, Joseph A.Bioorganic & Medicinal Chemistry Letters (2013), 23 (24), 6585-6587CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)The discovery of lead compd. 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray anal. The results of distribution, metab. and pharmacokinetics were reported. Compd. 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 μM), highly selective, and safe in in vitro and in vivo assays.
- 140Sato, K.; Kunitomo, Y.; Kasai, Y.; Utsumi, S.; Suetake, I.; Tajima, S.; Ichikawa, S.; Matsuda, A. Mechanism-Based Inhibitor of DNA Cytosine-5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2-Amino-4-Halopyridine-C-Nucleoside. ChemBioChem 2018, 19 (8), 865– 872, DOI: 10.1002/cbic.201700688[Crossref], [PubMed], [CAS], Google Scholar140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXktlyksLw%253D&md5=73265c9833d45b2931daf7ef0a251916Mechanism-Based Inhibitor of DNA Cytosine-5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2-Amino-4-Halopyridine-C-NucleosideSato, Kousuke; Kunitomo, Yuma; Kasai, Yukiko; Utsumi, Shohei; Suetake, Isao; Tajima, Shoji; Ichikawa, Satoshi; Matsuda, AkiraChemBioChem (2018), 19 (8), 865-872CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)In chromatin, 5-methylcytosine (mC), which represents the fifth nucleobase in genomic DNA, plays a role as an inducer of epigenetic changes. Tumor cells exhibit aberrant DNA methylation patterns, and inhibition of human DNA cytosine-5 methyltransferase (DNMT), which is responsible for generating mC in CpG sequences, is an effective strategy to treat various cancers. Here, we describe the design, synthesis, and evaluation of the properties of 2-amino-4-halopyridine-C-nucleosides (dXP) and oligodeoxyribonucleotides (ODNs) contg. dXP as a novel mechanism-based inhibitor of DNMTs. The designed ODN contg. XPpG forms a complex with DNMTs by covalent bonding through a nucleophilic arom. substitution (SNAr) reaction, and its cell proliferation activity is investigated. This study suggests that dXP in a CpG sequence of DNA could serve as a potential nucleic acid drug lead in cancer chemotherapy and a useful chem. probe for studies of epigenetics. Our mol. design using a SNAr reaction would be useful for DNMTs and other protein-DNA interactions.
- 141Kasai, Y.; Sato, K.; Utsumi, S.; Ichikawa, S. Improvement of SNAr Reaction Rate by an Electron-Withdrawing Group in the Crosslinking of DNA Cytosine-5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor. ChemBioChem 2018, 19 (17), 1866– 1872, DOI: 10.1002/cbic.201800244[Crossref], [PubMed], [CAS], Google Scholar141https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlWqurnN&md5=73faed58b2396d901a27c49b2aa13af3Improvement of SNAr Reaction Rate by an Electron-Withdrawing Group in the Crosslinking of DNA Cytosine-5 Methyltransferase by a Covalent Oligodeoxyribonucleotide InhibitorKasai, Yukiko; Sato, Kousuke; Utsumi, Shohei; Ichikawa, SatoshiChemBioChem (2018), 19 (17), 1866-1872CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)DNA cytosine 5-methyltransferase (DNMT) catalyzes methylation at the C5 position of the cytosine residues in the CpG sequence. Aberrant DNA methylation patterns are found in cancer cells. Therefore, inhibition of human DNMT is an effective strategy for treating various cancers. The inhibitors of DNMT have an electron-deficient nucleobase because this group facilitates attack by the catalytic Cys residue in DNMTs. Recently, we reported the synthesis and properties of mechanism-based modified nucleosides, 2-amino-4-halopyridine-C-nucleosides (dXP), as inhibitors of DNMT. To develop a more efficient inhibitor of DNMT for oligonucleotide therapeutics, oligodeoxyribonucleotides (ODNs) contg. other nucleoside analogs, which react more quickly with DNMT, are needed. Herein, we describe the design, synthesis, and evaluation of the properties of 2-amino-3-cyano-4-halopyridine-C-nucleosides (dXPCN) and ODNs contg. dXPCN, as more reactive inhibitors of DNMTs. Nucleophilic arom. substitution (SNAr) of the designed nucleosides, dXPCN, was faster than that of dXP, and the ODN contg. dXPCN effectively formed a complex with DNMTs. This study suggests that the incorporation of an electron-withdrawing group would be an effective method to increase reactivity toward the nucleophile of the DNMTs, while maintaining high specificity.
- 142Gianatassio, R.; Lopchuk, J. M.; Wang, J.; Pan, C.-M.; Malins, L. R.; Prieto, L.; Brandt, T. A.; Collins, M. R.; Gallego, G. M.; Sach, N. W.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Amination. Science 2016, 351 (6270), 241– 246, DOI: 10.1126/science.aad6252[Crossref], [PubMed], [CAS], Google Scholar142https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlvVCjuw%253D%253D&md5=868e44945c04c09881ac0d2516be23aeStrain-release aminationGianatassio, Ryan; Lopchuk, Justin M.; Wang, Jie; Pan, Chung-Mao; Malins, Lara R.; Prieto, Liher; Brandt, Thomas A.; Collins, Michael R.; Gallego, Gary M.; Sach, Neal W.; Spangler, Jillian E.; Zhu, Huichin; Zhu, Jinjiang; Baran, Phil S.Science (Washington, DC, United States) (2016), 351 (6270), 241-246CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)To optimize drug candidates, modern medicinal chemists are increasingly turning to an unconventional structural motif: small, strained ring systems. However, the difficulty of introducing substituents such as bicyclo[1.1.1]pentanes, azetidines, or cyclobutanes often outweighs the challenge of synthesizing the parent scaffold itself. Thus, there is an urgent need for general methods to rapidly and directly append such groups onto core scaffolds. Here we report a general strategy to harness the embedded potential energy of effectively spring-loaded C-C and C-N bonds with the most oft-encountered nucleophiles in pharmaceutical chem., amines. Strain-release amination can diversify a range of substrates with a multitude of desirable bioisosteres at both the early and late stages of a synthesis. The technique has also been applied to peptide labeling and bioconjugation.
- 143Lopchuk, J. M.; Fjelbye, K.; Kawamata, Y.; Malins, L. R.; Pan, C.-M.; Gianatassio, R.; Wang, J.; Prieto, L.; Bradow, J.; Brandt, T. A.; Collins, M. R.; Elleraas, J.; Ewanicki, J.; Farrell, W.; Fadeyi, O. O.; Gallego, G. M.; Mousseau, J. J.; Oliver, R.; Sach, N. W.; Smith, J. K.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Heteroatom Functionalization: Development, Scope, and Stereospecificity. J. Am. Chem. Soc. 2017, 139 (8), 3209– 3226, DOI: 10.1021/jacs.6b13229[ACS Full Text], [CAS], Google Scholar143https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVykur0%253D&md5=a7bd930dfd87427f5b8c64cb16fdbbe9Strain-Release Heteroatom Functionalization: Development, Scope, and StereospecificityLopchuk, Justin M.; Fjelbye, Kasper; Kawamata, Yu; Malins, Lara R.; Pan, Chung-Mao; Gianatassio, Ryan; Wang, Jie; Prieto, Liher; Bradow, James; Brandt, Thomas A.; Collins, Michael R.; Elleraas, Jeff; Ewanicki, Jason; Farrell, William; Fadeyi, Olugbeminiyi O.; Gallego, Gary M.; Mousseau, James J.; Oliver, Robert; Sach, Neal W.; Smith, Jason K.; Spangler, Jillian E.; Zhu, Huichin; Zhu, Jinjiang; Baran, Phil S.Journal of the American Chemical Society (2017), 139 (8), 3209-3226CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Driven by the ever-increasing pace of drug discovery and the need to push the boundaries of unexplored chem. space, medicinal chemists are routinely turning to unusual strained bioisosteres such as bicyclo[1.1.1]pentane, azetidine, and cyclobutane to modify their lead compds. Too often, however, the difficulty of installing these fragments surpasses the challenges posed even by the construction of the parent drug scaffold. This full account describes the development and application of a general strategy where spring-loaded, strained C-C and C-N bonds react with amines to allow for the "any-stage" installation of small, strained ring systems. In addn. to the functionalization of small building blocks and late-stage intermediates, the methodol. has been applied to bioconjugation and peptide labeling. For the first time, the stereospecific strain-release "cyclopentylation" of amines, alcs., thiols, carboxylic acids, and other heteroatoms is introduced. This report describes the development, synthesis, scope of reaction, bioconjugation, and synthetic comparisons of four new chiral "cyclopentylation" reagents.
- 144Semmler, K.; Szeimies, G.; Belzner, J. Tetracyclo[5.1.0.01,6.02,7]Octane, a [1.1.1]Propellane Derivative, and a New Route to the Parent Hydrocarbon. J. Am. Chem. Soc. 1985, 107 (22), 6410– 6411, DOI: 10.1021/ja00308a053[ACS Full Text], [CAS], Google Scholar144https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlslOqurc%253D&md5=a3846ab7c1f01f056f62e0a5e42a0076Tetracyclo[5.1.0.01,6.02,7]octane, a [1.1.1]propellane derivative, and a new route to the parent hydrocarbonSemmler, Klaus; Szeimies, Guenter; Belzner, JohannesJournal of the American Chemical Society (1985), 107 (22), 6410-11CODEN: JACSAT; ISSN:0002-7863.The title compd. (I) was prepd. from tricyclo[4.1.0.02,7]heptane. Carbenation (CHBr3) of (ClCH2)2C:CBr2, then cyclization with BuLi gave propellane (II).
- 145Haag, B.; Mosrin, M.; Ila, H.; Malakhov, V.; Knochel, P. Regio- and Chemoselective Metalation of Arenes and Heteroarenes Using Hindered Metal Amide Bases. Angew. Chem., Int. Ed. 2011, 50 (42), 9794– 9824, DOI: 10.1002/anie.201101960[Crossref], [CAS], Google Scholar145https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1Cjs7jI&md5=d8ed72b4eb7c627230c658547eb030f4Regio- and Chemoselective Metalation of Arenes and Heteroarenes Using Hindered Metal Amide BasesHaag, Benjamin; Mosrin, Marc; Ila, Hiriyakkanavar; Malakhov, Vladimir; Knochel, PaulAngewandte Chemie, International Edition (2011), 50 (42), 9794-9824CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The prepn. of highly functionalized organometallic compds. can be achieved by direct C-H activation of a broad range of unsatd. substrates using lithium chloride solubilized 2,2,6,6-tetramethylpiperidide bases (TMPnMXmp LiCl). These are excellent reagents for converting a wide range of arom. and heterocyclic substrates into valuable organometallic reagents with broad applications in org. synthesis.
- 146Wishart, D. S.; Feunang, Y. D.; Guo, A. C.; Lo, E. J.; Marcu, A.; Grant, J. R.; Sajed, T.; Johnson, D.; Li, C.; Sayeeda, Z.; Assempour, N.; Iynkkaran, I.; Liu, Y.; Maciejewski, A.; Gale, N.; Wilson, A.; Chin, L.; Cummings, R.; Le, D.; Pon, A.; Knox, C.; Wilson, M. DrugBank 5.0: A Major Update to the DrugBank Database for 2018. Nucleic Acids Res. 2018, 46 (D1), D1074– D1082, DOI: 10.1093/nar/gkx1037[Crossref], [PubMed], [CAS], Google Scholar146https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlGisbvI&md5=986b28c7ea546596a26dd3ba38f05feeDrugBank 5.0: a major update to the DrugBank database for 2018Wishart, David S.; Feunang, Yannick D.; Guo, An C.; Lo, Elvis J.; Marcu, Ana; Grant, Jason R.; Sajed, Tanvir; Johnson, Daniel; Li, Carin; Sayeeda, Zinat; Assempour, Nazanin; Iynkkaran, Ithayavani; Liu, Yifeng; Maciejewski, Adam; Gale, Nicola; Wilson, Alex; Chin, Lucy; Cummings, Ryan; Le, Diana; Pon, Allison; Knox, Craig; Wilson, MichaelNucleic Acids Research (2018), 46 (D1), D1074-D1082CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)DrugBank is a web-enabled database contg. comprehensivemol. information about drugs, their mechanisms, their interactions and their targets. First described in 2006, Drug- Bank has continued to evolve over the past 12 years in response to marked improvements to web stds. and changing needs for drug research and development. This year's update, DrugBank 5.0, represents the most significant upgrade to the database in more than 10 years. In many cases, existing data content has grown by 100% or more over the last update. For instance, the total no. of investigational drugs in the database has grown by almost 300%, the no. of drug-drug interactions has grown by nearly 600% and the no. of SNP-assocd. drug effects has grown more than 3000%. Significant improvements have been made to the quantity, quality and consistency of drug indications, drug binding data as well as drug-drug and drug-food interactions. A great deal of brand new data have also been added to DrugBank 5.0. This includes information on the influence of hundreds of drugs on metabolite levels (pharmacometabolomics), gene expression levels (pharmacotranscriptomics) and protein expression levels (pharmacoproteomics). New data have also been added on the status of hundreds of newdrug clin. trials and existing drug repurposing trials. Many other important improvements in the content, interface and performance of the DrugBank website have been made and these should greatly enhance its ease of use, utility and potential applications in many areas of pharmacol. research, pharmaceutical science and drug education.
- 147Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Applications of Fluorine in Medicinal Chemistry. J. Med. Chem. 2015, 58 (21), 8315– 8359, DOI: 10.1021/acs.jmedchem.5b00258[ACS Full Text], [CAS], Google Scholar147https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1ajs7%252FK&md5=9995829a94a8c0b8d9fb0d21bdfd5a1dApplications of Fluorine in Medicinal ChemistryGillis, Eric P.; Eastman, Kyle J.; Hill, Matthew D.; Donnelly, David J.; Meanwell, Nicholas A.Journal of Medicinal Chemistry (2015), 58 (21), 8315-8359CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review with meta-anal. The role of fluorine in drug design and development is expanding rapidly as we learn more about the unique properties assocd. with this unusual element and how to deploy it with greater sophistication. The judicious introduction of fluorine into a mol. can productively influence conformation, pKa, intrinsic potency, membrane permeability, metabolic pathways, and pharmacokinetic properties. In addn., 18F has been established as a useful positron emitting isotope for use with in vivo imaging technol. that potentially has extensive application in drug discovery and development, often limited only by convenient synthetic accessibility to labeled compds. The wide ranging applications of fluorine in drug design are providing a strong stimulus for the development of new synthetic methodologies that allow more facile access to a wide range of fluorinated compds. In this review, we provide an update on the effects of the strategic incorporation of fluorine in drug mols. and applications in positron emission tomog.
- 148Meanwell, N. A. Fluorine and Fluorinated Motifs in the Design and Application of Bioisosteres for Drug Design. J. Med. Chem. 2018, 61 (14), 5822– 5880, DOI: 10.1021/acs.jmedchem.7b01788[ACS Full Text], [CAS], Google Scholar148https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2qu7w%253D&md5=2d0ce3326c7ff932da8d7d26972ced14Fluorine and Fluorinated Motifs in the Design and Application of Bioisosteres for Drug DesignMeanwell, Nicholas A.Journal of Medicinal Chemistry (2018), 61 (14), 5822-5880CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The electronic properties and relatively small size of fluorine endow it with considerable versatility as a bioisostere and it has found application as a substitute for lone pairs of electrons, the hydrogen atom, and the Me group while also acting as a functional mimetic of the carbonyl, carbinol, andnitrile moieties. In this context, fluorine substitution can influence the potency, conformation, metab., membrane permeability, and P-gp recognition of a mol. and temper inhibition of the hERG channel by basic amines. However, as a consequence of the unique properties of fluorine, it features prominently in the design of higher order structural metaphors that are more esoteric in their conception and which reflect a more sophisticated mol. construction that broadens biol. mimesis. In this Perspective, applications of fluorine in the construction of bioisosteric elements designed to enhance the in vitro and in vivo properties of a mol. are summarized.
- 149Grishin, N. V.; Osterman, A. L.; Brooks, H. B.; Phillips, M. A.; Goldsmith, E. J. X-Ray Structure of Ornithine Decarboxylase from Trypanosoma Brucei: The Native Structure and the Structure in Complex with α-Difluoromethylornithine. Biochemistry 1999, 38 (46), 15174– 15184, DOI: 10.1021/bi9915115[ACS Full Text], [CAS], Google Scholar149https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXmvFCrurs%253D&md5=83ae6756feca1823788b041bbf331542X-ray structure of ornithine decarboxylase from Trypanosoma brucei: The native structure and the structure in complex with α-difluoromethylornithineGrishin, Nick V.; Osterman, Andrei L.; Brooks, Harold B.; Phillips, Margaret A.; Goldsmith, Elizabeth J.Biochemistry (1999), 38 (46), 15174-15184CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by T. brucei. One of the currently used drugs, α-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO was detd. by x-ray crystallog. to 2.0 Å resoln. The protein crystd. in space group P21 (a = 66.8, b = 154.5, c = 77.1 Å, and β = 90.58°), with 2 dimers per asym. unit. The initial phasing was done by mol. replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also detd. to 2.9 Å resoln. by mol. replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC was a β/α-barrel, and the C-terminal domain of ODC was a modified Greek key β-barrel. In comparison to structurally related alanine racemase, the 2 domains were rotated 27° relative to each other. In addn., 2 of the β-strands in the C-terminal domain had exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface were formed primarily by the C-terminal domains, which interacted through 6 arom. rings that formed stacking interactions across the domain boundary. The PLP-binding site was formed by the C-termini of β-strands and loops in the β/α-barrel. In the native structure, Lys-69 formed a Schiff base with PLP. In both structures, the phosphate of PLP was bound between the 7th and 8th strands forming interactions with Arg-277 and a Gly loop (residues 235-237). The pyridine N atom of PLP interacted with Glu-274. DFMO formed a Schiff base with PLP and was covalently attached to Cys-360. It was bound at the dimer interface and the δ-C amino group of DFMO was positioned between Asp-361 of one subunit and Asp-332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 had rotated 145° toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes were obsd. upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.
- 150Eckstein, J. W.; Foster, P. G.; Finer-Moore, J.; Wataya, Y.; Santi, D. V. Mechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2’-Deoxyuridine 5′-Monophosphate. Biochemistry 1994, 33 (50), 15086– 15094, DOI: 10.1021/bi00254a018[ACS Full Text], [CAS], Google Scholar150https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmvFGrt70%253D&md5=f82c290ac5cb6d16115a3517f283477aMechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2'-deoxyuridine 5'-MonophosphateEckstein, Jens W.; Foster, Paul G.; Finer-Moore, Janet; Wataya, Yusuke; Santi, Daniel V.Biochemistry (1994), 33 (50), 15086-94CODEN: BICHAW; ISSN:0006-2960.Thymidylate synthase (TS) from Lactobacillus casei is inhibited by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF3dUMP). CF3dUMP binds to the active site of TS in the absence of CH2H4folate, and attack of the catalytic nucleophile cysteine 198 at C6 of the pyrimidine leads to activation of the trifluoromethyl group and release of fluoride ion. Subsequently, the activated heterocycle reacts with a nucleophile of the enzyme to form a moderately stable covalent complex. Proteolytic digestion of TS treated with [2'-3H]CF3dUMP, followed by sequencing of the labeled peptides, revealed that tyrosine 146 and cysteine 198 are covalently bound to the inhibitor in the enzyme-inhibitor complex. The presence of dithiothreitol (DTT) or β-mercaptoethanol resulted in the breakdown of the covalent complex, and products from the breakdown of the complex were isolated and characterized. The three-dimensional structure of the enzyme-inhibitor complex was detd. by x-ray crystallog., clearly demonstrating covalent attachment of the nucleotide to tyrosine 146. A chem. reaction mechanism for the inhibition of TS by CF3dUMP is presented that is consistent with the kinetic, biochem., and structural results.
- 151Cohen, M. S.; Zhang, C.; Shokat, K. M.; Taunton, J. Structural Bioinformatics-Based Design of Selective, Irreversible Kinase Inhibitors. Science 2005, 308 (5726), 1318– 1321, DOI: 10.1126/science1108367[Crossref], [PubMed], [CAS], Google Scholar151https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXks1Ciurs%253D&md5=1364444dada4d1d46510aa0212428952Structural Bioinformatics-Based Design of Selective, Irreversible Kinase InhibitorsCohen, Michael S.; Zhang, Chao; Shokat, Kevan M.; Taunton, JackScience (Washington, DC, United States) (2005), 308 (5726), 1318-1321CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The active sites of 491 human protein kinase domains are highly conserved, which makes the design of selective inhibitors a formidable challenge. We used a structural bioinformatics approach to identify two selectivity filters, a threonine and a cysteine, at defined positions in the active site of p90 ribosomal protein S6 kinase (RSK). A fluoromethylketone inhibitor, designed to exploit both selectivity filters, potently and selectively inactivated RSK1 and RSK2 in mammalian cells. Kinases with only one selectivity filter were resistant to the inhibitor, yet they became sensitized after genetic introduction of the second selectivity filter. Thus, two amino acids that distinguish RSK from other protein kinases are sufficient to confer inhibitor sensitivity.
- 152Cohen, M. S.; Hadjivassiliou, H.; Taunton, J. A Clickable Inhibitor Reveals Context-Dependent Autoactivation of P90 RSK. Nat. Chem. Biol. 2007, 3 (3), 156– 160, DOI: 10.1038/nchembio859[Crossref], [PubMed], [CAS], Google Scholar152https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhslWgsL4%253D&md5=68f7da1c274678e884ea71d1078b092cA clickable inhibitor reveals context-dependent autoactivation of p90 RSKCohen, Michael S.; Hadjivassiliou, Haralambos; Taunton, JackNature Chemical Biology (2007), 3 (3), 156-160CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)P90 ribosomal protein S6 kinases (RSKs) integrate upstream signals through two catalytic domains. Autophosphorylation of Ser386 by the regulatory C-terminal kinase domain (CTD) is thought to be essential for activation of the N-terminal kinase domain (NTD), which phosphorylates multiple downstream targets. We recently reported fmk, an irreversible inhibitor of the CTD of RSK1 and RSK2. Here we describe fmk-pa, a propargylamine variant that has improved cellular potency and a 'clickable' tag for assessing the extent and selectivity of covalent RSK modification. Copper-catalyzed conjugation of an azidoalkyl reporter (the click reaction) revealed that fmk-pa achieves selective and saturable modification of endogenous RSK1 and RSK2 in mammalian cells. Satg. concns. of fmk-pa inhibited Ser386 phosphorylation and downstream signaling in response to phorbol ester stimulation, but had no effect on RSK activation by lipopolysaccharide. RSK autoactivation by the CTD is therefore context dependent, which suggests that NTD and CTD inhibitors should have distinct physiol. effects.
- 153Carroll, F. A. Perspectives on Structure and Mechanism in Organic Chemistry, 2nd ed.; Wiley-VCH: Weinheim, 2014.
- 154Shaik, S. S. The .Alpha.- and .Beta.-Carbon Substituent Effect on SN2 Reactivity. A Valence-Bond Approach. J. Am. Chem. Soc. 1983, 105 (13), 4359– 4367, DOI: 10.1021/ja00351a039[ACS Full Text], [CAS], Google Scholar154https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXkt1WlsrY%253D&md5=36aa6ec687f8a91cbf37372449cfaf05The α- and β-carbon substituent effect on SN2 reactivity. A valence-bond approachShaik, Sason S.Journal of the American Chemical Society (1983), 105 (13), 4359-67CODEN: JACSAT; ISSN:0002-7863.α- And β-C substituent effect, on SN2 reactivity and reactivity-selectivity, is discussed by using a (previously described) correlation diagram model of the SN2 reaction. The reaction barrier (E) is a fraction (f) of the energy gap (IN: - ARX) between the 2 curves which intersect to yield the reaction profile. IN: Is the ionization potential of the nucleophile (N:) and ARX is the electron affinity of the substrate (RX). The f which enters the activation barrier depends inter alia on the degree of delocalization of the three-electron bonds, e.g., (R···X)-. The more delocalized the three-electron bond, the larger the f. Reactivity trends arise from the interplay between the electron transfer aspect (IN: - ARX) and the bond-interchange aspect (e.g., the degree of delocalization of the three-electron bonds) of the SN2 transformation. α-Halo substitution (on R) delocalizes the three-electron bond and effects a small improvement in the acceptor ability of the substrate; it therefore slows down SN2 reactivity. The largest delocalization is effected when the α-substituent(s) is (are) identical with the leaving group. In these cases, the strongest rate retardation is obsd. π-Acceptor α-substituents improve the substrate acceptor ability markedly without greatly delocalizing the three-electron bonds. Therefore, these substituents will enhance reactivity but mainly toward powerful nucleophiles. The effects of other α-substituents (e.g., MeO, Ph, SiR3, etc.) and β-substituents (e.g., F, Cl, Br, RO, etc.) are also discussed. The reactivity reversals often reported in the literature are suggested to be manifestations of the gap-slope interplay. The reactivity of alkyl halides is extensively examd.
- 155Lundell, N.; Schreitmüller, T. Sample Preparation for Peptide Mapping— A Pharmaceutical Quality-Control Perspective. Anal. Biochem. 1999, 266 (1), 31– 47, DOI: 10.1006/abio.1998.2919[Crossref], [PubMed], [CAS], Google Scholar155https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXisV2ltQ%253D%253D&md5=0d20ad326def5e9bdf5df9d1e7ee1961Sample preparation for peptide mapping - a pharmaceutical quality-control perspectiveLundell, Niklas; Schreitmuller, ThomasAnalytical Biochemistry (1999), 266 (1), 31-47CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)A review with 116 refs. In quality control of therapeutic proteins peptide mapping is used for confirmation of primary structure and detection of posttranslational modifications. The demands put on the exptl. procedure are therefore different than in the case of detn. of an unknown protein structure. It is here recognized that a peptide-mapping method for quality control of proteins should be inert (not induce or revert modifications), general, robust, and allow a high sample throughput. The steps prior to the sepn. of the generated peptides are identified as crucial for meeting these demands. This includes denaturation, redn., alkylation, buffer exchange, solubilization, and digestion. A crit. review of the literature regarding these steps is presented. Relevant options in all steps are exptl. evaluated. Novel approaches are developed for many of the steps. The result is a sample prepn. procedure that essentially meets the stated demands. (c) 1999 Academic Press.
- 156Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B. D.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F. Quantitative Reactivity Profiling Predicts Functional Cysteines in Proteomes. Nature 2010, 468 (7325), 790– 795, DOI: 10.1038/nature09472[Crossref], [PubMed], [CAS], Google Scholar156https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVGhsr%252FI&md5=277e436e7a8f4dfff0b548bce52ac40dQuantitative reactivity profiling predicts functional cysteines in proteomesWeerapana, Eranthie; Wang, Chu; Simon, Gabriel M.; Richter, Florian; Khare, Sagar; Dillon, Myles B. D.; Bachovchin, Daniel A.; Mowen, Kerri; Baker, David; Cravatt, Benjamin F.Nature (London, United Kingdom) (2010), 468 (7325), 790-795CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochem. functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quant. the intrinsic reactivity of cysteine residues en masse directly in native biol. systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulfur protein biogenesis. We also demonstrate that quant. reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.
- 157Weerapana, E.; Simon, G. M.; Cravatt, B. F. Disparate Proteome Reactivity Profiles of Carbon Electrophiles. Nat. Chem. Biol. 2008, 4 (7), 405– 407, DOI: 10.1038/nchembio.91[Crossref], [PubMed], [CAS], Google Scholar157https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnt1ejtL8%253D&md5=69eb0ae589c50f46a8695dc6731366e2Disparate proteome reactivity profiles of carbon electrophilesWeerapana, Eranthie; Simon, Gabriel M.; Cravatt, Benjamin F.Nature Chemical Biology (2008), 4 (7), 405-407CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Insights into the proteome reactivity of electrophiles are crucial for designing activity-based probes for enzymes lacking cognate affinity labels. Here, the authors show that different classes of carbon electrophiles exhibit markedly distinct amino acid labeling profiles in proteomes, ranging from selective reactivity with cysteine to adducts with several amino acids. These data specify electrophilic chemotypes with restricted and permissive reactivity profiles to guide the design of next-generation functional proteomics probes.
- 158Lonsdale, R.; Burgess, J.; Colclough, N.; Davies, N. L.; Lenz, E. M.; Orton, A. L.; Ward, R. A. Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity. J. Chem. Inf. Model. 2017, 57 (12), 3124– 3137, DOI: 10.1021/acs.jcim.7b00553[ACS Full Text], [CAS], Google Scholar158https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsl2gsLjF&md5=722e83699c58dee1009b87c8c31bdeccExpanding the Armory: Predicting and Tuning Covalent Warhead ReactivityLonsdale, Richard; Burgess, Jonathan; Colclough, Nicola; Davies, Nichola L.; Lenz, Eva M.; Orton, Alexandra L.; Ward, Richard A.Journal of Chemical Information and Modeling (2017), 57 (12), 3124-3137CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)Targeted covalent inhibition is an established approach for increasing the potency and selectivity of potential drug candidates, as well as identifying potent and selective tool compds. for target validation studies. It is evident that identification of reversible recognition elements is essential for selective covalent inhibition, but this must also be achieved with the appropriate level of inherent reactivity of the reactive functionality (or "warhead"). Structural changes that increase or decrease warhead reactivity, guided by methods to predict the effect of those changes, have the potential to tune warhead reactivity and negate issues related to potency and/or toxicity. The half-life to adduct formation with glutathione (GSH t1/2) is a useful assay for measuring the reactivity of cysteine-targeting covalent warheads but is limited to synthesized mols. In this manuscript the authors assess the ability of several exptl. and computational approaches to predict GSH t1/2 for a range of cysteine targeting warheads, including a novel method based on pKa. Furthermore, matched mol. pairs anal. has been performed against the internal compd. collection, revealing structure-activity relationships between a selection of different covalent warheads. These observations and methods of prediction will be valuable in the design of new covalent inhibitors with desired levels of reactivity.
- 159Allimuthu, D.; Adams, D. J. 2-Chloropropionamide As a Low-Reactivity Electrophile for Irreversible Small-Molecule Probe Identification. ACS Chem. Biol. 2017, 12 (8), 2124– 2131, DOI: 10.1021/acschembio.7b00424[ACS Full Text], [CAS], Google Scholar159https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpvVGqsrw%253D&md5=39b4794ebb55d3314a7bac5b04ddadc72-Chloropropionamide As a Low-Reactivity Electrophile for Irreversible Small-Molecule Probe IdentificationAllimuthu, Dharmaraja; Adams, Drew J.ACS Chemical Biology (2017), 12 (8), 2124-2131CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Resurgent interest in covalent target engagement in drug discovery has demonstrated that small mols. contg. weakly reactive electrophiles can be safe and effective therapies. Several recently FDA-approved drugs feature an acrylamide functionality to selectively engage cysteine side chains of kinases (Ibrutinib, Afatinib, and Neratinib). Addnl. electrophilic functionalities whose reactivity is compatible with highly selective target engagement and in vivo application could open new avenues in covalent small mol. discovery. Here the authors report the synthesis and evaluation of a library of small mols. contg. the 2-chloropropionamide functionality, which the authors demonstrate is less reactive than typical acrylamide electrophiles. Although many library members do not appear to label proteins in cells, the authors identified S-CW3554 as selectively labeling protein disulfide isomerase and inhibiting its enzymic activity. Subsequent profiling of the library against five diverse cancer cell lines showed unique cytotoxicity for S-CW3554 I in cells derived from multiple myeloma, a cancer recently reported to be sensitive to PDI inhibition. The novel PDI inhibitor highlights the potential of 2-chloropropionamides as weak and stereochem.-tunable electrophiles for covalent drug discovery.
- 160Steinmetz, C. G.; Xie, P.; Weiner, H.; Hurley, T. D. Structure of Mitochondrial Aldehyde Dehydrogenase: The Genetic Component of Ethanol Aversion. Structure 1997, 5 (5), 701– 711, DOI: 10.1016/S0969-2126(97)00224-4[Crossref], [PubMed], [CAS], Google Scholar160https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjvFamtLw%253D&md5=c66dc959c97a17f68490b588d9d5419aStructure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversionSteinmetz, Curtis G.; Xie, Peiguang; Weiner, Henry; Hurley, Thomas D.Structure (London) (1997), 5 (5), 701-711CODEN: STRUE6; ISSN:0969-2126. (Current Biology)The single genetic factor most strongly correlated with reduced alc. consumption and incidence of alcoholism is a naturally occurring variant of mitochondrial aldehyde dehydrogenase (ALDH2). This variant contains a glutamate to lysine substitution at position 487 (E487K). The E487K variant of ALDH2 is found in approx. 50% of the Asian population, and is assocd. with a phenotypic loss of ALDH2 activity in both heterozygotes and homozygotes. ALDH2-deficient individuals exhibit an averse response to ethanol consumption, which is probably caused by elevated levels of blood acetaldehyde. The structure of ALDH2 is important for the elucidation of its catalytic mechanism, to gain a clear understanding of the contribution of ALDH2 to the genetic component of alcoholism and for the development of specific ALDH2 inhibitors as potential drugs for use in the treatment of alcoholism. The x-ray structure of bovine ALDH2 has been solved to 2.65 Å in its free form and to 2.75 Å in a complex with NAD+. The enzyme structure contains three domains: two dinucleotide-binding domains and a small three-stranded β-sheet domain, which is involved in subunit interactions in this tetrameric enzyme. The E487K mutation occurs in this small oligomerization domain and is located at a key interface between subunits immediately below the active site of another monomer. The active site of ALDH2 is divided into two halves by the nicotinamide ring of NAD+. Adjacent to the A-side (Pro-R) of the nicotinamide ring is a cluster of three cysteines (Cys301, Cys302 and Cys303) and adjacent to the B-side (Pro-S) are Thr244, Glu268, Glu476 and an ordered water mol. bound to Thr244 and Glu476. Although there is a recognizable Rossmann-type fold, the coenzyme-binding region of ALDH2 binds NAD+ in a manner not seen in other NAD+-binding enzymes. The positions of the residues near the nicotinamide ring of NAD+ suggest a chem. mechanism whereby Glu268 functions as a general base through a bound water mol. The sidechain amide nitrogen of Asn169 and the peptide nitrogen of Cys302 are in position to stabilize the oxyanion present in the tetrahedral transition state prior to hydride transfer. The functional importance of residue Glu487 now appears to be due to indirect interactions of this residue with the substrate-binding site via Arg264 and Arg475.
- 161Karala, A.-R.; Lappi, A.-K.; Ruddock, L. W. Modulation of an Active-Site Cysteine PKa Allows PDI to Act as a Catalyst of Both Disulfide Bond Formation and Isomerization. J. Mol. Biol. 2010, 396 (4), 883– 892, DOI: 10.1016/j.jmb.2009.12.014[Crossref], [PubMed], [CAS], Google Scholar161https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitValtr4%253D&md5=7fb917968538e6ed0f15277e254ef224Modulation of an Active-Site Cysteine pKa Allows PDI to Act as a Catalyst of both Disulfide Bond Formation and IsomerizationKarala, Anna-Riikka; Lappi, Anna-Kaisa; Ruddock, Lloyd W.Journal of Molecular Biology (2010), 396 (4), 883-892CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Protein disulfide isomerase (PDI) plays a central role in disulfide bond formation in the endoplasmic reticulum. It is implicated both in disulfide bond formation and in disulfide bond redn. and isomerization. To be an efficient catalyst of all three reactions requires complex mechanisms. These include mechanisms to modulate the pKa values of the active-site cysteines of PDI. Here, the role of arginine 120 in modulating the pKa values of these cysteines was examd. It was found that arginine 120 plays a significant role in modulating the pKa of the C-terminal active-site cysteine in the a domain of PDI and plays a role in detg. the reactivity of the N-terminal active-site cysteine but not via direct modulation of its pKa. Mutation of arginine 120 and the corresponding residue, arginine 461, in the a' domain severely reduces the ability of PDI to catalyze disulfide bond formation and redn. but enhances the ability to catalyze disulfide bond isomerization due to the formation of more stable PDI-substrate mixed disulfides. These results suggest that the modulation of pKa of the C-terminal active cysteine by the movement of the side chain of these arginine residues into the active-site locales has evolved to allow PDI to efficiently catalyze both oxidn. and isomerization reactions.
- 162Wang, C.; Abegg, D.; Hoch, D. G.; Adibekian, A. Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT. Angew. Chem., Int. Ed. 2016, 55 (8), 2911– 2915, DOI: 10.1002/anie.201511301[Crossref], [CAS], Google Scholar162https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1Ojt74%253D&md5=37ddd0a42f0d2c87207fd8c27f3f9496Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMTWang, Chao; Abegg, Daniel; Hoch, Dominic G.; Adibekian, AlexanderAngewandte Chemie, International Edition (2016), 55 (8), 2911-2915CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors present a novel chem. scaffold for cysteine-reactive covalent inhibitors. Chloromethyl triazoles (CMTs) are readily accessed in only two chem. steps, thus enabling the rapid optimization of the pharmacol. properties of these inhibitors. The authors demonstrate the tunability of the CMTs towards a specific biol. target by synthesizing I as the first potent nonpseudosubstrate inhibitor of the O6-alkylguanine DNA methyltransferase (MGMT), a protein of major clin. significance for the treatment of several severe cancer forms. Using quant. proteomics profiling techniques, the authors show that I exhibits a high degree of selectivity towards MGMT. Finally, the authors validate the effectiveness of the MGMT inhibitor in combination with the DNA alkylating drug temozolomide in breast and colon cancer cells by fluorescence imaging and a cell-viability assay. The results may open a new avenue towards the development of a clin. approved MGMT inhibitor.
- 163Boren, B. C.; Narayan, S.; Rasmussen, L. K.; Zhang, L.; Zhao, H.; Lin, Z.; Jia, G.; Fokin, V. V. Ruthenium-Catalyzed Azide–Alkyne Cycloaddition: Scope and Mechanism. J. Am. Chem. Soc. 2008, 130 (28), 8923– 8930, DOI: 10.1021/ja0749993[ACS Full Text], [CAS], Google Scholar163https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXns1Wjtr8%253D&md5=8b5ba21060ad9d6984bba3e2587aa44dRuthenium-Catalyzed Azide-Alkyne Cycloaddition: Scope and MechanismBoren, Brant C.; Narayan, Sridhar; Rasmussen, Lars K.; Zhang, Li; Zhao, Haitao; Lin, Zhenyang; Jia, Guochen; Fokin, Valery V.Journal of the American Chemical Society (2008), 130 (28), 8923-8930CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The catalytic activity of ruthenium(II) complexes in azide-alkyne cycloaddns. were evaluated. The [Cp*RuCl] complexes, such as Cp*RuCl(PPh3)2, Cp*RuCl(COD), and Cp*RuCl(NBD), were among the most effective catalysts. In the presence of catalytic Cp*RuCl(PPh3)2 or Cp*RuCl(COD), primary and secondary azides react with a broad range of terminal alkynes contg. a range of functionalities selectively producing 1,5-disubstituted 1,2,3-triazoles; tertiary azides were significantly less reactive. Both complexes also promote the cycloaddn. reactions of org. azides with internal alkynes, providing access to fully-substituted 1,2,3-triazoles. The ruthenium-catalyzed azide-alkyne cycloaddn. (RuAAC) appears to proceed via oxidative coupling of the azide and alkyne reactants to give a six-membered ruthenacycle intermediate, in which the first new carbon-nitrogen bond is formed between the more electroneg. carbon of the alkyne and the terminal, electrophilic nitrogen of the azide. This step is followed by reductive elimination, which forms the triazole product. DFT calcns. support this mechanistic proposal and indicate that the reductive elimination step is rate-detg.
- 164Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Epoxide Electrophiles as Activity-Dependent Cysteine Protease Profiling and Discovery Tools. Chem. Biol. 2000, 7 (8), 569– 581, DOI: 10.1016/S1074-5521(00)00014-4[Crossref], [PubMed], [CAS], Google Scholar164https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnt1WnsbY%253D&md5=4bfc0e299524198ec324c96c8981baf7Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery toolsGreenbaum, Doron; Medzihradszky, Katalin F.; Burlingame, Alma; Bogyo, MatthewChemistry & Biology (2000), 7 (8), 569-581CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Science Ltd.)Background: Anal. of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly detg. activities of an entire class of enzymes on a genome-wide scale would be of great utility. Results: We have engineered chem. probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes contg. a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivs. was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular exts. Finally, the affinity tag of DCG-04 allowed purifn. of modified proteases and identification by mass spectrometry. Conclusions: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixts. These probes were used to det. relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purifn./identification of targets.
- 165Willems, L. I.; Jiang, J.; Li, K.-Y.; Witte, M. D.; Kallemeijn, W. W.; Beenakker, T. J. N.; Schröder, S. P.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. From Covalent Glycosidase Inhibitors to Activity-Based Glycosidase Probes. Chem. - Eur. J. 2014, 20 (35), 10864– 10872, DOI: 10.1002/chem.201404014[Crossref], [PubMed], [CAS], Google Scholar165https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlWmtLjO&md5=45059a66dcfd0a2895da38996d0a7be1From Covalent Glycosidase Inhibitors to Activity-Based Glycosidase ProbesWillems, Lianne I.; Jiang, Jianbing; Li, Kah-Yee; Witte, Martin D.; Kallemeijn, Wouter W.; Beenakker, Thomas J. N.; Schroeder, Sybrin P.; Aerts, Johannes M. F. G.; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; Overkleeft, Hermen S.Chemistry - A European Journal (2014), 20 (35), 10864-10872CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Activity-based protein profiling has emerged as a powerful discovery tool in chem. biol. and medicinal chem. research. Success of activity-based protein profiling hinges on the presence of compds. that can covalently and irreversibly bind to enzymes, do so selectively in the context of complex biol. samples, and subsequently report on the selected pool of proteins. Such tagged mols. featuring an electrophilic trap, termed activity-based probes, have been developed with most success for serine hydrolases and various protease families (serine proteases, cysteine proteases, proteasomes). This concept presents the current progress and future directions in the design of activity-based probes targeting retaining glycosidases, enzymes that employ a double displacement mechanism in the hydrolysis of glycosidic bonds with overall retention. In contrast to inverting glycosidases, retaining glycosidases form a covalent intermediate with their substrates during the catalytic process and are therefore amenable to activity-based protein profiling studies.
- 166Adams, B. T.; Niccoli, S.; Chowdhury, M. A.; Esarik, A. N. K.; Lees, S. J.; Rempel, B. P.; Phenix, C. P. N -Alkylated Aziridines Are Easily-Prepared, Potent, Specific and Cell-Permeable Covalent Inhibitors of Human β-Glucocerebrosidase. Chem. Commun. 2015, 51 (57), 11390– 11393, DOI: 10.1039/C5CC03828F[Crossref], [PubMed], [CAS], Google Scholar166https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVCmu7nI&md5=c5f088dbe0d16ac976f5070113e6f74bN-Alkylated aziridines are easily-prepared, potent, specific and cell-permeable covalent inhibitors of human β-glucocerebrosidaseAdams, B. T.; Niccoli, S.; Chowdhury, M. A.; Esarik, A. N. K.; Lees, S. J.; Rempel, B. P.; Phenix, C. P.Chemical Communications (Cambridge, United Kingdom) (2015), 51 (57), 11390-11393CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)β-Glucocerebrosidase deficiency leads to Gaucher disease and is a potential marker of Parkinson's disease. The authors have identified N-octyl conduritol aziridine as a potent and specific covalent inactivator of GBA1 in living cells. This compd. is a promising lead towards a positron emission tomog. probe intended to image GBA1 activity.
- 167Singh, G. S. Synthetic Aziridines in Medicinal Chemistry: A Mini-Review. Mini-Rev. Med. Chem. 2016, 16, 892– 904, DOI: 10.2174/1389557515666150709122244[Crossref], [PubMed], [CAS], Google Scholar167https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XptVWltrw%253D&md5=b538dbb714d03d160cf85a59938db843Synthetic Aziridines in Medicinal Chemistry: A Mini-ReviewSingh, Girija S.Mini-Reviews in Medicinal Chemistry (2016), 16 (11), 892-904CODEN: MMCIAE; ISSN:1389-5575. (Bentham Science Publishers Ltd.)Azaheterocyclic compds. are well-known to have diverse types of biol. activity. Among them, azacyclopropanes, commonly referred as aziridines, occupy a prominent place in synthetic org. and medicinal chem. due to its occurrence in natural resources, complexity involved in synthesis due to ring-strain, building blocks in org. synthesis, and its biol. properties. Several novel compds. contg. aziridine ring have been designed and synthesized recently by medicinal chemists for evaluating their biol. profile. A no. of compds. are reported as cysteine protease inhibitors, antibacterial, antifungal, anticancer, antileishmanial, and antimalarial agents. This review article summarizes the biol. activity of such compds. The prepn. of such compds. is also described.
- 168Pitscheider, M.; Mäusbacher, N.; Sieber, S. A. Antibiotic Activity and Target Discovery of Three-Membered Natural Product-Derived Heterocycles in Pathogenic Bacteria. Chem. Sci. 2012, 3, 2035– 2041, DOI: 10.1039/c2sc20290e[Crossref], [CAS], Google Scholar168https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xnt12ru7c%253D&md5=2f89f4792f2ddbd48366cca7beea0904Antibiotic activity and target discovery of three-membered natural product-derived heterocycles in pathogenic bacteriaPitscheider, Maximilian; Maeusbacher, Nina; Sieber, Stephan A.Chemical Science (2012), 3 (6), 2035-2041CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Oxirane, thiirane and aziridine scaffolds represent core motifs in many natural products that usually exhibit their bioactivity by a covalent modification of essential active site residues via a nucleophilic ring opening reaction. Synthesis of these three-membered heterocyclic activity based probes (ABP) was accomplished by robust std. procedures and complemented by a similar set of Michael acceptor systems. Subsequent reactivity studies against the full complement of enzymes in several bacteria revealed an individual fine-tuned affinity and reactivity of each probe that depended on the decoration as well as the heteroatom, resp. Out of many interesting probe-target pairs, we identified thiirane-based compds. that were capable of labeling essential enzymes in S. aureus and L. monocytogenes. Corresponding to the labeling pattern we obsd. an antibiotic effect of these compds. against the clin. relevant pathogens L. monocytogenes as well as multiresistant S. aureus (MRSA).
- 169Lee, M.; Ikejiri, M.; Klimpel, D.; Toth, M.; Espahbodi, M.; Hesek, D.; Forbes, C.; Kumarasiri, M.; Noll, B. C.; Chang, M.; Mobashery, S. Structure–Activity Relationship for Thiirane-Based Gelatinase Inhibitors. ACS Med. Chem. Lett. 2012, 3 (6), 490– 495, DOI: 10.1021/ml300050b[ACS Full Text], [CAS], Google Scholar169https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmsVWgurg%253D&md5=dd16caf1a6f18e07eebfeb53a0a69dd9Structure-Activity Relationship for Thiirane-Based Gelatinase InhibitorsLee, Mijoon; Ikejiri, Masahiro; Klimpel, Dennis; Toth, Marta; Espahbodi, Mana; Hesek, Dusan; Forbes, Christopher; Kumarasiri, Malika; Noll, Bruce C.; Chang, Mayland; Mobashery, ShahriarACS Medicinal Chemistry Letters (2012), 3 (6), 490-495CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)An extensive structure-activity relationship study with the template of 2-(4-phenoxyphenylsulfonylmethyl)thiirane (I), a potent and highly selective inhibitor for human gelatinases, is reported herein. Syntheses of 65 new analogs, each in multistep processes, allowed for exploration of key structural components of the mol. template. This study reveals that the presence of the sulfonylmethylthiirane and the phenoxyphenyl group were important for gelatinase inhibition. However, para- and some meta-substitutions of the terminal Ph ring enhanced inhibitory activity and led to improve metabolic stability. This agrees with the result from metab. studies with compd. I that the primary route of biotransformation is oxidn., mainly at the para position of the Ph ring and the α position of the sulfonyl group in the aliph. side chain.
- 170Harshbarger, W.; Miller, C.; Diedrich, C.; Sacchettini, J. Crystal Structure of the Human 20S Proteasome in Complex with Carfilzomib. Structure 2015, 23 (2), 418– 424, DOI: 10.1016/j.str.2014.11.017[Crossref], [PubMed], [CAS], Google Scholar170https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtF2msbg%253D&md5=0249966770dad6c25b7844fb1cea59b1Crystal Structure of the Human 20S Proteasome in Complex with CarfilzomibHarshbarger, Wayne; Miller, Chase; Diedrich, Chandler; Sacchettini, JamesStructure (Oxford, United Kingdom) (2015), 23 (2), 418-424CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)Proteasome inhibition is highly effective as a treatment for multiple myeloma, and recently carfilzomib was granted US FDA approval for the treatment of relapsed and refractory multiple myeloma. Here, we report the X-ray crystal structure of the human constitutive 20S proteasome with and without carfilzomib bound at 2.9 and 2.6 Å, resp. Our data indicate that the S3 and S4 binding pockets play a pivotal role in carfilzomib's selectivity for chymotrypsin-like sites. Structural comparison with the mouse immunoproteasome crystal structure reveals amino acid substitutions that explain carfilzomib's slight preference for chymotrypsin-like subunits of constitutive proteasomes. In addn., comparison of the human proteasome:carfilzomib complex with the mouse proteasome:PR-957 complex reveals new details that explain why PR-957 is selective for immunoproteasomes. Together, the data presented here support the design of inhibitors for either constitutive or immunoproteasomes, with implications for the treatment of cancers as well as autoimmune and neurodegenerative diseases.
- 171Falagas, M. E.; Vouloumanou, E. K.; Samonis, G.; Vardakas, K. Z. Fosfomycin. Clin. Microbiol. Rev. 2016, 29 (2), 321– 347, DOI: 10.1128/CMR.00068-15[Crossref], [PubMed], [CAS], Google Scholar171https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmsVyhsbY%253D&md5=56a1033535467b306574356bd943fa4bFosfomycinFalagas, Matthew E.; Vouloumanou, Evridiki K.; Samonis, George; Vardakasa, Konstantinos Z.Clinical Microbiology Reviews (2016), 29 (2), 321-347CODEN: CMIREX; ISSN:1098-6618. (American Society for Microbiology)The treatment of bacterial infections suffers from two major problems: spread of multidrug-resistant (MDR) or extensively drug-resistant (XDR) pathogens and lack of development of new antibiotics active against such MDR and XDR bacteria. As a result, physicians have turned to older antibiotics, such as polymyxins, tetracyclines, and aminoglycosides. Lately, due to development of resistance to these agents, fosfomycin has gained attention, as it has remained active against both Gram-pos. and Gram-neg. MDR and XDR bacteria. New data of higher quality have become available, and several issues were clarified further. In this review, we summarize the available fosfomycin data regarding pharmacokinetic and pharmacodynamic properties, the in vitro activity against susceptible and antibiotic-resistant bacteria, mechanisms of resistance and development of resistance during treatment, synergy and antagonism with other antibiotics, clin. effectiveness, and adverse events. Issues that need to be studied further are also discussed.
- 172Kim, D. H.; Lees, W. J.; Kempsell, K. E.; Lane, W. S.; Duncan, K.; Walsh, C. T. Characterization of a Cys115 to Asp Substitution in the Escherichia Coli Cell Wall Biosynthetic Enzyme UDP-GlcNAc Enolpyruvyl Transferase (MurA) That Confers Resistance to Inactivation by the Antibiotic Fosfomycin. Biochemistry 1996, 35 (15), 4923– 4928, DOI: 10.1021/bi952937w[ACS Full Text], [CAS], Google Scholar172https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XhvVCls7w%253D&md5=49606bbdd8809592a5a2c78ece07e4ddCharacterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc Enolpyruvyl Transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycinKim, Dennis H.; Lees, Watson J.; Kempsell, Karen E.; Lane, William S.; Duncan, Kenneth; Walsh, Christopher T.Biochemistry (1996), 35 (15), 4923-8CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The antibiotic fosfomycin inhibits bacterial cell wall biosynthesis by inactivation of UDP-GlcNAc enolpyruvyl transferase (MurA). Prior work has established that Cys115 of Escherichia coli and Enterobacter cloacae MurA is the active site nucleophile alkylated by fosfomycin and implicated this residue in the formation of a covalent phospholactyl-enzyme adduct derived from the substrate, phosphoenolpyruvate (PEP). On the basis of sequencing information from a putative MurA homolog from Mycobacterium tuberculosis, the authors generated a C115D mutant of E. coli MurA that was highly active but fully resistant to time-dependent inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the obsd. reversible competitive inhibition vs. PEP. In contrast to the broad pH-independent behavior of wild-type (WT) MurA, C115D mutant activity titrated across the pH range examd. (pH 5.5-9) with an apparent pKa ∼6, with kcatC115D ranging from ∼10kcatWT at pH 5.5 to <0.1kcatWT at pH 9.0. Km(PEP)C115D was relatively const. in the pH range examd. and increased ∼100-fold relative to Km(PEP)WT. A fosfomycin-resistant C115E mutant with ∼1% activity of the C115D mutant was found to follow a pH dependence similar to that obsd. for C115D MurA. The contrasting pH dependences of WT and C115D MurA were also obsd. in the reaction with the pseudosubstrate, (Z)-3-fluorophosphoenolpyruvate, strongly suggesting a role for Cys/Asp115 as the general acid in the protonation of C-3 of PEP during MurA-catalyzed enol ether transfer. The difference in nucleophilicity between the carboxylate side chains of Asp115 and Glu115 and the thiolate group of Cys115 suggests that covalent enzyme adduct formation is not required for catalytic turnover and, furthermore, provides a chem. rationale for the resistance of the C115D and C115E mutants to fosfomycin inactivation.
- 173Eschenburg, S.; Priestman, M.; Schönbrunn, E. Evidence That the Fosfomycin Target Cys115 in UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Is Essential for Product Release. J. Biol. Chem. 2005, 280 (5), 3757– 3763, DOI: 10.1074/jbc.M411325200[Crossref], [PubMed], [CAS], Google Scholar173https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXovVGmsw%253D%253D&md5=6142eb1dd85ae7d39ef2345c415473b6Evidence that the fosfomycin target Cys115 in UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is essential for product releaseEschenburg, Susanne; Priestman, Melanie; Schoenbrunn, ErnstJournal of Biological Chemistry (2005), 280 (5), 3757-3763CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)MurA (UDP-N-acetylglucosamine enolpyruvyl transferase, EC 2.5.1.7) is an essential enzyme in the biosynthesis of the peptidoglycan layer of the bacterial cell. It provides an attractive template for the design of novel antibiotic drugs and is the target of the naturally occurring antibiotic fosfomycin, which covalently attaches to Cys115 in the active site of the enzyme. Mutations of Cys115 to Asp exist in pathogens such as Mycobacteria or Chlamydia rendering these organisms resistant to fosfomycin. Thus, there is a need for the elucidation of the role of this cysteine in the MurA reaction. We detd. the x-ray structure of the C115S mutant of Enterobacter cloacae MurA, which was crystd. in the presence of the substrates of MurA. The structure depicts the product state of the enzyme with enolpyruvyl-UDP-N-acetylglucosamine (EP-UNAG) and inorg. phosphate (Pi) trapped in the active site. Kinetic anal. revealed that the Cys-to-Ser mutation results in an enzyme that appears to perform a single turnover of the reaction. Opposing the common view of Cys115 as a key residue in the chem. reaction of enolpyruvyl transfer, we now conclude that the wild-type cysteine is essential for product release only. On the basis of a detailed comparison of the product state with the intermediate state and an unliganded state of MurA, we propose that dissocn. of the products is an ordered event with inorg. phosphate leaving first. Phosphate departure appears to trigger a suite of conformational changes, which finally leads to opening of the two-domain structure of MurA and the release of the second product enolpyruvyl-UDP-N-acetylglucosamine.
- 174Porter, N. J.; Christianson, D. W. Binding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8. ACS Chem. Biol. 2017, 12 (9), 2281– 2286, DOI: 10.1021/acschembio.7b00330[ACS Full Text], [CAS], Google Scholar174https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtl2gsrjE&md5=18fbcd74c854bf9183e3d67074338aaeBinding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8Porter, Nicholas J.; Christianson, David W.ACS Chemical Biology (2017), 12 (9), 2281-2286CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Trapoxin A is a microbial cyclic tetrapeptide that is an essentially irreversible inhibitor of class I histone deacetylases (HDACs). The inhibitory warhead is the α,β-epoxyketone side-chain of (2S,9S)-2-amino-8-oxo-9,10-epoxydecanoic acid (L-Aoe), which mimics the side-chain of the HDAC substrate, acetyl-L-lysine. Here, we report the crystal structure of the HDAC8-trapoxin A complex at 1.24 Å resoln., revealing that the ketone moiety of L-Aoe undergoes nucleophilic attack to form a Zn-bound tetrahedral gem-diolate that mimics the tetrahedral intermediate and its flanking transition states in catalysis. Mass spectrometry, activity measurements, and isothermal titrn. calorimetry confirmed that trapoxin A binds tightly (Kd = 3 ± 1 nM) and did not covalently modify the enzyme, so that the epoxide moiety of L-Aoe remained intact. Comparison of the HDAC8-trapoxin A complex with the HDAC6-HC toxin complex provided new insight regarding the inhibitory potency of L-Aoe-contg. natural products against class I and class II HDACs.
- 175Lopus, M.; Smiyun, G.; Miller, H.; Oroudjev, E.; Wilson, L.; Jordan, M. A. Mechanism of Action of Ixabepilone and Its Interactions with the ΒIII-Tubulin Isotype. Cancer Chemother. Pharmacol. 2015, 76 (5), 1013– 1024, DOI: 10.1007/s00280-015-2863-z[Crossref], [PubMed], [CAS], Google Scholar175https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsF2htrvM&md5=0509fbdac680a6e951f27762cc909ef6Mechanism of action of ixabepilone and its interactions with the βIII-tubulin isotypeLopus, Manu; Smiyun, Greg; Miller, Herb; Oroudjev, Emin; Wilson, Leslie; Jordan, Mary AnnCancer Chemotherapy and Pharmacology (2015), 76 (5), 1013-1024CODEN: CCPHDZ; ISSN:0344-5704. (Springer)Ixabepilone (Ixempra, BMS-247550), a semisynthetic analog of epothilone B, is a microtubule-targeted drug in clin. use for treatment of metastatic or locally advanced breast cancer. Ixabepilone's binding and mechanism of action on microtubules and their dynamics, as well as its interactions with isotypically altered microtubules, both in vitro and in tumor cells, have not been described. Microtubules are dynamic polymers of the protein tubulin that function in mitosis, intracellular transport, cell proliferation, and migration. They continually undergo dynamic instability, periods of slow growth and rapid shortening that are crucial to these cell functions. We detd. ixabepilone's microtubule binding and polymn. effects in vitro and also detd. its effects on inhibition of dynamic instability in vitro and in cells, both with and without removal of the βIII isotype of tubulin. The βIII isotype of tubulin is assocd. with drug resistance and tumor aggressivity. We found that removal (in vitro) and knockdown (in cells) of βIII-tubulin led to increased inhibition of microtubule dynamic instability by ixabepilone. Depletion of βIII-tubulin from MCF7 human breast cancer cells also induced increased mitotic arrest by ixabepilone. Thus, βIII-tubulin expression suppresses the antitumor effects of ixabepilone, indicating that increased βIII-tubulin may be an important contributor to the development of resistance to ixabepilone.
- 176Carmi, C.; Cavazzoni, A.; Vezzosi, S.; Bordi, F.; Vacondio, F.; Silva, C.; Rivara, S.; Lodola, A.; Alfieri, R. R.; La Monica, S.; Galetti, M.; Ardizzoni, A.; Petronini, P. G.; Mor, M. Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the Cysteine-Trap Portion. J. Med. Chem. 2010, 53 (5), 2038– 2050, DOI: 10.1021/jm901558p[ACS Full Text], [CAS], Google Scholar176https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhvFOrsbw%253D&md5=5231cae7a553cb1c282e3d09d6901551Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the Cysteine-Trap PortionCarmi, Caterina; Cavazzoni, Andrea; Vezzosi, Stefano; Bordi, Fabrizio; Vacondio, Federica; Silva, Claudia; Rivara, Silvia; Lodola, Alessio; Alfieri, Roberta R.; La Monica, Silvia; Galetti, Maricla; Ardizzoni, Andrea; Petronini, Pier Giorgio; Mor, MarcoJournal of Medicinal Chemistry (2010), 53 (5), 2038-2050CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Irreversible EGFR inhibitors can circumvent acquired resistance to first-generation reversible, ATP-competitive inhibitors in the treatment of non-small-cell lung cancer. They contain both a driver group, which assures target recognition, and a warhead, generally an acrylamide or propargylamide fragment that binds covalently to Cys797 within the kinase domain of EGFR. We performed a systematic exploration of the role for the warhead group, introducing different cysteine-trapping fragments at position 6 of a traditional 4-anilinoquinazoline scaffold. We found that different reactive groups, including epoxyamides (compds. 3-6) and phenoxyacetamides (compds. 7-9), were able to irreversibly inhibit EGFR. In particular, at significant lower concns. than gefitinib (1), (2R,3R)-N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(piperidin-1-ylmethyl)oxirane-2-carboxamide (6) inhibited EGFR autophosphorylation and downstream signaling pathways, suppressed proliferation, and induced apoptosis in gefitinib-resistant NSCLC H1975 cells, harboring the T790M mutation in EGFR.
- 177Klüter, S.; Simard, J. R.; Rode, H. B.; Grütter, C.; Pawar, V.; Raaijmakers, H. C. A.; Barf, T. A.; Rabiller, M.; van Otterlo, W. A. L.; Rauh, D. Characterization of Irreversible Kinase Inhibitors by Directly Detecting Covalent Bond Formation: A Tool for Dissecting Kinase Drug Resistance. ChemBioChem 2010, 11 (18), 2557– 2566, DOI: 10.1002/cbic.201000352[Crossref], [PubMed], [CAS], Google Scholar177https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFahsLrE&md5=5f48cde54e6bad28b4d4f9d47a4e3e3eCharacterization of Irreversible Kinase Inhibitors by Directly Detecting Covalent Bond Formation: A Tool for Dissecting Kinase Drug ResistanceKlueter, Sabine; Simard, Jeffrey R.; Rode, Haridas B.; Gruetter, Christian; Pawar, Vijaykumar; Raaijmakers, Hans C. A.; Barf, Tjeerd A.; Rabiller, Matthias; van Otterlo, Willem A. L.; Rauh, DanielChemBioChem (2010), 11 (18), 2557-2566CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Targeting protein kinases in cancer therapy with irreversible small-mol. inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-assocd. drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biol. and biochem. studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants.
- 178Gehringer, M.; Forster, M.; Laufer, S. A. Solution-Phase Parallel Synthesis of Ruxolitinib-Derived Janus Kinase Inhibitors via Copper-Catalyzed Azide–Alkyne Cycloaddition. ACS Comb. Sci. 2015, 17 (1), 5– 10, DOI: 10.1021/co500122h[ACS Full Text], [CAS], Google Scholar178https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFCisbnI&md5=5d2999fedb2dfef332c2e860a9e376e4Solution-Phase Parallel Synthesis of Ruxolitinib-Derived Janus Kinase Inhibitors via Copper-Catalyzed Azide-Alkyne CycloadditionGehringer, Matthias; Forster, Michael; Laufer, Stefan A.ACS Combinatorial Science (2015), 17 (1), 5-10CODEN: ACSCCC; ISSN:2156-8944. (American Chemical Society)A soln.-phase parallel synthesis of triazole-derived ruxolitinib analogs, e.g. I, was developed in the current study. The method employs copper-catalyzed azide-alkyne cycloaddn. to build up the central triazole template. Product isolation by pptn. and centrifugation is straightforward and yields high purity compds. suited for biol. profiling. A simple protocol for accessing the terminal alkyne precursors in high yields was established and a library of ruxolitinib-like triazoles featuring diverse functional groups was prepd. In addn., a model for the binding mode of ruxolitinib to Janus kinase (JAK) 2 is proposed. In contrast to previous models, the pose explains the compd. selectivity for JAK1/JAK2 and is in accordance with published structure-activity data. On this basis, a structure-based design hypothesis for inverting the selectivity profile of ruxolitinib is deduced. Application of this strategy identified a moderately potent JAK3 inhibitor (35 nM) with high selectivity against other JAKs, potentially exploiting a covalent binding mode.
- 179Forster, M.; Chaikuad, A.; Bauer, S. M.; Holstein, J.; Robers, M. B.; Corona, C. R.; Gehringer, M.; Pfaffenrot, E.; Ghoreschi, K.; Knapp, S.; Laufer, S. A. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket. Cell Chem. Biol. 2016, 23 (11), 1335– 1340, DOI: 10.1016/j.chembiol.2016.10.008[Crossref], [PubMed], [CAS], Google Scholar179https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVeqsLrJ&md5=348c5b5fbfa91fd02fa21d984620ea16Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding PocketForster, Michael; Chaikuad, Apirat; Bauer, Silke M.; Holstein, Julia; Robers, Matthew B.; Corona, Cesear R.; Gehringer, Matthias; Pfaffenrot, Ellen; Ghoreschi, Kamran; Knapp, Stefan; Laufer, Stefan A.Cell Chemical Biology (2016), 23 (11), 1335-1340CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Janus kinases (JAKs) are a family of cytoplasmatic tyrosine kinases that are attractive targets for the development of anti-inflammatory drugs given their roles in cytokine signaling. One question regarding JAKs and their inhibitors that remains under intensive debate is whether JAK inhibitors should be isoform selective. Since JAK3 functions are restricted to immune cells, an isoform-selective inhibitor for JAK3 could be esp. valuable to achieve clin. more useful and precise effects. However, the high degree of structural conservation makes isoform-selective targeting a challenging task. Here, we present picomolar inhibitors with unprecedented kinome-wide selectivity for JAK3. Selectivity was achieved by concurrent covalent reversible targeting of a JAK3-specific cysteine residue and a ligand-induced binding pocket. We confirmed that in vitro activity and selectivity translate well into the cellular environment and suggest that our inhibitors are powerful tools to elucidate JAK3-specific functions.
- 180Rempel, B. P.; Withers, S. G. Covalent Inhibitors of Glycosidases and Their Applications in Biochemistry and Biology. Glycobiology 2008, 18 (8), 570– 586, DOI: 10.1093/glycob/cwn041[Crossref], [PubMed], [CAS], Google Scholar180https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXoslGlsL4%253D&md5=d0a41272cc33a51eaeeb086229ea5789Covalent inhibitors of glycosidases and their applications in biochemistry and biologyRempel, Brian P.; Withers, Stephen G.Glycobiology (2008), 18 (8), 570-586CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)A review. Glycoside hydrolases are important enzymes in a no. of essential biol. processes. Irreversible inhibitors of this class of enzyme have attracted interest as probes of both structure and function. Here, the authors discuss some of the compds. used to covalently modify glycosidases, their use in residue identification, structural and mechanistic investigations, and finally their applications, both in vitro and in vivo, to complex biol. systems.
- 181Povirk, L. F.; Shuker, D. E. DNA Damage and Mutagenesis Induced by Nitrogen Mustards. Mutat. Res., Rev. Genet. Toxicol. 1994, 318 (3), 205– 226, DOI: 10.1016/0165-1110(94)90015-9[Crossref], [PubMed], [CAS], Google Scholar181https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXitlaktbo%253D&md5=e29f751b42550ae503639e6b2fc0fdd1DNA damage and mutagenesis induced by nitrogen mustardsPovirk, Lawrence F.; Shuker, David E.Mutation Research, Reviews in Genetic Toxicology (1994), 318 (3), 205-26CODEN: MRRTEP; ISSN:0165-1110. (Elsevier B.V.)A review with many refs. The nitrogen mustards are bifunctional alkylating agents which, although used extensively in cancer chemotherapy, are themselves highly carcinogenic. All nitrogen mustards induce monofunctional guanine-N7 adducts, as well as interstrand N7-N7 crosslinks involving the two guanines in GNC·GNC (5'→3'/5'→3') sequences. In addn., the arom. mustards melphalan and chlorambucil also induce substantial alkylation at adenine N3, while cyclophosphamide forms phosphotriesters with relatively high frequency. Nitrogen mustards are genotoxic in virtually every assay, and produce a wide array of mutations, including base substitutions at both G·C and A·T base pairs, intragenic as well as multilocus deletions, and chromosomal rearrangements. Mutational spectra generated by these agents in various model systems vary widely, and no single lesion has been implicated as being primarily responsible for mustard-induced mutagenesis. On the contrary, adducts of both adenine and guanine, and monofunctional as well as bifunctional adducts, appear to be involved. Further, it is still not known which types of mutation are responsible for mustard-induced cancers, since no genes have yet been identified which are consistently altered in these malignancies.
- 182McGregor, L. M.; Jenkins, M. L.; Kerwin, C.; Burke, J. E.; Shokat, K. M. Expanding the Scope of Electrophiles Capable of Targeting K-Ras Oncogenes. Biochemistry 2017, 56 (25), 3178– 3183, DOI: 10.1021/acs.biochem.7b00271[ACS Full Text], [CAS], Google Scholar182https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVGrsbnI&md5=0b25b65210248a4cad2916efba90ddaeExpanding the Scope of Electrophiles Capable of Targeting K-Ras OncogenesMcGregor, Lynn M.; Jenkins, Meredith L.; Kerwin, Caitlin; Burke, John E.; Shokat, Kevan M.Biochemistry (2017), 56 (25), 3178-3183CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)There is growing interest in reversible and irreversible covalent inhibitors that target noncatalytic amino acids in target proteins. With a goal of targeting oncogenic K-Ras variants (e.g., G12D) by expanding the types of amino acids that can be targeted by covalent inhibitors, we survey a set of electrophiles for their ability to label carboxylates. We functionalized an optimized ligand for the K-Ras switch II pocket with a set of electrophiles previously reported to react with carboxylates and characterized the ability of these compds. to react with model nucleophiles and oncogenic K-Ras proteins. Here, we report that aziridines and stabilized diazo groups preferentially react with free carboxylates over thiols. Although we did not identify a warhead that potently labels K-Ras G12D, we were able to study the interactions of many electrophiles with K-Ras, as most of the electrophiles rapidly label K-Ras G12C. We characterized the resulting complexes by crystallog., hydrogen/deuterium exchange, and differential scanning fluorometry. Our results both demonstrate the ability of a noncatalytic cysteine to react with a diverse set of electrophiles and emphasize the importance of proper spatial arrangements between a covalent inhibitor and its intended nucleophile. We hope that these results can expand the range of electrophiles and nucleophiles of use in covalent protein modulation.
- 183Ray, S.; Kreitler, D. F.; Gulick, A. M.; Murkin, A. S. The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition. ACS Chem. Biol. 2018, 13 (6), 1470– 1473, DOI: 10.1021/acschembio.8b00225[ACS Full Text], [CAS], Google Scholar183https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpsl2nsLc%253D&md5=d2dc8f255fd7e5f7537c57a0c5a3343eThe nitro group as a masked electrophile in covalent enzyme inhibitionRay, Sneha; Kreitler, Dale F.; Gulick, Andrew M.; Murkin, Andrew S.ACS Chemical Biology (2018), 13 (6), 1470-1473CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)We report the unprecedented reaction between a nitroalkane and an active-site cysteine residue to yield a thiohydroximate adduct. Structural and kinetic evidence suggests the nitro group is activated by conversion to its nitronic acid tautomer within the active site. The nitro group, therefore, shows promise as a masked electrophile in the design of covalent inhibitors targeting binding pockets with appropriately placed cysteine and general acid residues.
- 184Moynihan, M. M.; Murkin, A. S. Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate. Biochemistry 2014, 53 (1), 178– 187, DOI: 10.1021/bi401432t[ACS Full Text], [CAS], Google Scholar184https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFOiurvM&md5=07384a8e75df3a87db4ed9e0010b12bcCysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-NitropropionateMoynihan, Margaret M.; Murkin, Andrew S.Biochemistry (2014), 53 (1), 178-187CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Isocitrate lyase (ICL) catalyzes the reversible cleavage of isocitrate into succinate and glyoxylate. It is the first committed step in the glyoxylate cycle used by some organisms, including Mycobacterium tuberculosis, where it has been shown to be essential for cell survival during chronic infection. The pH-rate and pD-rate profiles measured in the direction of isocitrate synthesis revealed solvent kinetic isotope effects (KIEs) of 1.7 ± 0.4 for D2OV and 0.56 ± 0.07 for D2O(V/Ksuccinate). Whereas the D2OV is consistent with partially rate-limiting proton transfer during formation of the hydroxyl group of isocitrate, the large inverse D2O(V/Ksuccinate) indicates that substantially different kinetic parameters exist when the enzyme is satd. with succinate. Inhibition by 3-nitropropionate (3-NP), a succinate analog, was found to proceed through an unusual double slow-onset process featuring formation of a complex with a Ki of 3.3 ± 0.2 μM during the first minute, followed by formation of a final complex with a Ki* of 44 ± 10 nM over the course of several minutes to hours. Stopped-flow measurements during the first minute revealed an apparent solvent KIE of 0.40 ± 0.03 for assocn. and unity for dissocn. In contrast, itaconate, a succinate analog lacking an acidic α-proton, did not display slow-binding behavior and yielded a D2OKi of 1.0 ± 0.2. These results support a common mechanism for catalysis with succinate and inhibition by 3-NP featuring (1) an unfavorable prebinding isomerization of the active site Cys191-His193 pair to the thiolate-imidazolium form, a process that is favored in D2O, and (2) the transfer of a proton from succinate or 3-NP to Cys191. These findings also indicate that propionate-3-nitronate, which is the conjugate base of 3-NP and the "true inhibitor" of ICL, does not bind directly and must be generated enzymically.
- 185Krenske, E. H.; Petter, R. C.; Houk, K. N. Kinetics and Thermodynamics of Reversible Thiol Additions to Mono- and Diactivated Michael Acceptors: Implications for the Design of Drugs That Bind Covalently to Cysteines. J. Org. Chem. 2016, 81 (23), 11726– 11733, DOI: 10.1021/acs.joc.6b02188[ACS Full Text], [CAS], Google Scholar185https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhsl2gtbbK&md5=64cf60c3ab5b5778b28cbd0aed528d37Kinetics and Thermodynamics of Reversible Thiol Additions to Mono- and Diactivated Michael Acceptors: Implications for the Design of Drugs That Bind Covalently to CysteinesKrenske, Elizabeth H.; Petter, Russell C.; Houk, K. N.Journal of Organic Chemistry (2016), 81 (23), 11726-11733CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)Addns. of cysteine thiols to Michael acceptors underpin the mechanism of action of several covalent drugs (e.g., afatinib, osimertinib, ibrutinib, neratinib, and CC-292). Reversible Michael acceptors were reported in which an addnl. electron-withdrawing group was added at the α-carbon of a Michael acceptor. The authors have performed d. functional theory calcns. to det. why thiol addns. to these Michael acceptors are reversible. The α-EWG group stabilizes the anionic transition state and intermediate of the Michael addn., but less intuitively, it destabilizes the neutral adduct. This makes the reverse reaction (elimination) both faster and more thermodynamically favorable. For thiol addn. to be reversible, the Michael acceptor must also contain a suitable substituent on the β-carbon, such as an aryl or branched alkyl group. Computations explain how these structural elements contribute to reversibility and the ability to tune the binding affinities and the residence times of covalent inhibitors.
- 186Krishnan, S.; Miller, R. M.; Tian, B.; Mullins, R. D.; Jacobson, M. P.; Taunton, J. Design of Reversible, Cysteine-Targeted Michael Acceptors Guided by Kinetic and Computational Analysis. J. Am. Chem. Soc. 2014, 136 (36), 12624– 12630, DOI: 10.1021/ja505194w[ACS Full Text], [CAS], Google Scholar186https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVWrtbvK&md5=de052bac07c8920cb807c05f71f4f95dDesign of Reversible, Cysteine-Targeted Michael Acceptors Guided by Kinetic and Computational AnalysisKrishnan, Shyam; Miller, Rand M.; Tian, Boxue; Mullins, R. Dyche; Jacobson, Matthew P.; Taunton, JackJournal of the American Chemical Society (2014), 136 (36), 12624-12630CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacol. potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo β-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our exptl. and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.
- 187Smith, S.; Keul, M.; Engel, J.; Basu, D.; Eppmann, S.; Rauh, D. Characterization of Covalent-Reversible EGFR Inhibitors. ACS Omega 2017, 2 (4), 1563– 1575, DOI: 10.1021/acsomega.7b00157[ACS Full Text], [CAS], Google Scholar187https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmt1KqsLg%253D&md5=695eb8fbc519ba2fba3ff7d5296b4116Characterization of Covalent-Reversible EGFR InhibitorsSmith, Steven; Keul, Marina; Engel, Julian; Basu, Debjit; Eppmann, Simone; Rauh, DanielACS Omega (2017), 2 (4), 1563-1575CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)Within the spectrum of kinase inhibitors, covalent-reversible inhibitors (CRIs) provide a valuable alternative approach to classical covalent inhibitors. This special class of inhibitors can be optimized for an extended drug-target residence time. For CRIs, it was shown that the fast addn. of thiols to electron-deficient olefins leads to a covalent bond that can break reversibly under proteolytic conditions. Research groups are just beginning to include CRIs in their arsenal of compd. classes, and, with that, the understanding of this interesting set of chem. warheads is growing. However, systems to assess both characteristics of the covalent-reversible bond in a simple exptl. setting are sparse. Here, we have developed an efficient methodol. to characterize the covalent and reversible properties of CRIs and to investigate their potential in targeting clin. relevant variants of the receptor tyrosine kinase EGFR.
- 188Forster, M.; Chaikuad, A.; Dimitrov, T.; Döring, E.; Holstein, J.; Berger, B.-T.; Gehringer, M.; Ghoreschi, K.; Müller, S.; Knapp, S.; Laufer, S. A. Development, Optimization, and Structure–Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4-d]Pyrrolo[2,3-b]Pyridine Scaffold. J. Med. Chem. 2018, 61 (12), 5350– 5366, DOI: 10.1021/acs.jmedchem.8b00571[ACS Full Text], [CAS], Google Scholar188https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVGjsrbN&md5=8acdec137df746f854bbee75a10d5f70Development, Optimization, and Structure-Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4-d]pyrrolo[2,3-b]pyridine ScaffoldForster, Michael; Chaikuad, Apirat; Dimitrov, Teodor; Doering, Eva; Holstein, Julia; Berger, Benedict-Tilman; Gehringer, Matthias; Ghoreschi, Kamran; Mueller, Susanne; Knapp, Stefan; Laufer, Stefan A.Journal of Medicinal Chemistry (2018), 61 (12), 5350-5366CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Janus kinases are major drivers of immune signaling and have been the focus of anti-inflammatory drug discovery for more than a decade. Because of the invariable colocalization of JAK1 and JAK3 at cytokine receptors, the question if selective JAK3 inhibition is sufficient to effectively block downstream signaling has been highly controversial. Recently, we discovered the covalent-reversible JAK3 inhibitor FM-381 (23) featuring high isoform and kinome selectivity. Crystallog. revealed that this inhibitor induces an unprecedented binding pocket by interactions of a nitrile substituent with arginine residues in JAK3. Herein, we describe detailed structure-activity relationships necessary for induction of the arginine pocket and the impact of this structural change on potency, isoform selectivity, and efficacy in cellular models. Furthermore, we evaluated the stability of this novel inhibitor class in in vitro metabolic assays and were able to demonstrate an adequate stability of key compd. 23 for in vivo use.
- 189Cheung, S. T.; Miller, M. S.; Pacoma, R.; Roland, J.; Liu, J.; Schumacher, A. M.; Hsieh-Wilson, L. C. Discovery of a Small-Molecule Modulator of Glycosaminoglycan Sulfation. ACS Chem. Biol. 2017, 12 (12), 3126– 3133, DOI: 10.1021/acschembio.7b00885[ACS Full Text], [CAS], Google Scholar189https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslGrsbfE&md5=4626679495c584d0ce81daa64a920b7aDiscovery of a Small-Molecule Modulator of Glycosaminoglycan SulfationCheung, Sheldon T.; Miller, Michelle S.; Pacoma, Reynand; Roland, Jason; Liu, Jian; Schumacher, Andrew M.; Hsieh-Wilson, Linda C.ACS Chemical Biology (2017), 12 (12), 3126-3133CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Glycosaminoglycans (GAGs) play crit. roles in diverse processes ranging from viral infection to neuroregeneration. Their regiospecific sulfation patterns, which are generated by sulfotransferases, are key structural determinants that underlie their biol. activity. Small-mol. modulators of these sulfotransferases could serve as powerful tools for understanding the physiol. functions of GAGs, as well as potential therapeutic leads for human diseases. Here, the authors report the development of the first cell-permeable, small-mol. inhibitor selective for GAG sulfotransferases, which was obtained using a high-throughput screen targeted against Chst15, the sulfotransferase responsible for biosynthesis of chondroitin sulfate-E (CS-E). The authors demonstrate that the mol. specifically inhibits GAG sulfotransferases in vitro, decreases CS-E and overall sulfation levels on cell-surface and secreted chondroitin sulfate proteoglycans (CSPGs), and reverses CSPG-mediated inhibition of axonal growth. These studies pave the way toward a new set of pharmacol. tools for interrogating GAG sulfation-dependent processes and may represent a novel therapeutic approach for neuroregeneration.
- 190Cully, M. Rational Drug Design: Tuning Kinase Inhibitor Residence Time. Nat. Rev. Drug Discovery 2015, 14, 457, DOI: 10.1038/nrd4673[Crossref], [PubMed], [CAS], Google Scholar190https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFeju7nI&md5=3bcab237493059803552dafd0e4fc6e5Rational drug design Tuning kinase inhibitor residence timeCully, MeganNature Reviews Drug Discovery (2015), 14 (7), 457CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)There is no expanded citation for this reference.
- 191Langrish, C. L.; Bradshaw, J. M.; Owens, T. D.; Campbell, R. L.; Francesco, M. R.; Karr, D. E.; Murray, S. K.; Quesenberry, R. C.; Smith, P. F.; Taylor, M. D.; Zhu, J.; Nunn, P. A.; Gourlay, S. G. PRN1008, a Reversible Covalent BTK Inhibitor in Clinical Development for Immune Thrombocytopenic Purpura. Blood 2017, 130, 1052
- 192Masjedizadeh, M. R.; Gourlay, S. Salts and Solid Form of a Btk Inhibitor. WO2015127310, August 28, 2015.
- 193Holm, K. J.; Spencer, C. M. Entacapone. Drugs 1999, 58 (1), 159– 177, DOI: 10.2165/00003495-199958010-00017[Crossref], [PubMed], [CAS], Google Scholar193https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXltFOju7Y%253D&md5=3285c98f7cdf43918bad3fded4a39bc5Entacapone: a review of its use in Parkinson's diseaseHolm, Kristin J.; Spencer, Caroline M.Drugs (1999), 58 (1), 159-177CODEN: DRUGAY; ISSN:0012-6667. (Adis International Ltd.)A review with 58 refs. Entacapone is a potent and specific peripheral catechol-O-methyltransferase (COMT) inhibitor. It has been shown to improve the clin. benefits of levodopa plus an arom. L-amino acid decarboxylase inhibitor (AADC) when given to patients with Parkinson's disease and end-of-dose deterioration in the response to levodopa (the "wearing off" phenomenon). The efficacy of entacapone is currently being assessed in patients with stable Parkinson's disease. In 2 well conducted trials of 6 mo' duration and smaller short term studies, treatment with entacapone (200mg with each dose of levodopa/AADC inhibitor) was assocd. with significant increases in daily "on" time and decreases in "off" time. Changes in Unified Parkinson's Disease Rating Scale (UPDRS) scores concurred with changes in "on" and "off" times: entacapone improved total, activities of daily living and motor function scores, but it had no effect on mentation scores. Entacapone also provided benefits when given with controlled release levodopa/AADC inhibitor or with std. levodopa/AADC inhibitor and selegiline in small trials. Dopaminergic events, including dyskinesia and nausea, are among the most common events with entacapone, and are related to the drug's ability to potentiate the effects of levodopa. Diarrhea, abdominal pain, constipation and urine discoloration are the most common nondopaminergic events, although the latter event is the only one to occur consistently more frequently with entacapone than with placebo. However, adverse events of any type infrequently led to study discontinuation. The efficacy and tolerability of entacapone administered with levodopa/AADC inhibitor have not yet been compared with those of other strategies for the treatment of Parkinson's disease. However, once the decision to initiate levodopa therapy has been made, studies generally support the use of entacapone as an adjunct to levodopa in patients with Parkinson's disease and the "wearing off" phenomenon.
- 194Mendgen, T.; Steuer, C.; Klein, C. D. Privileged Scaffolds or Promiscuous Binders: A Comparative Study on Rhodanines and Related Heterocycles in Medicinal Chemistry. J. Med. Chem. 2012, 55 (2), 743– 753, DOI: 10.1021/jm201243p[ACS Full Text], [CAS], Google Scholar194https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVGkt7nN&md5=6a94d097f44bdbaaec9a56fd6b371521Privileged Scaffolds or Promiscuous Binders: A Comparative Study on Rhodanines and Related Heterocycles in Medicinal ChemistryMendgen, Thomas; Steuer, Christian; Klein, Christian D.Journal of Medicinal Chemistry (2012), 55 (2), 743-753CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Rhodanines and related five-membered heterocycles with multiple heteroatoms have recently gained a reputation of being unselective compds. that appear as "frequent hitters" in screening campaigns and therefore have little value in drug discovery. However, this judgment appears to be based mostly on anecdotal evidence. Having identified various rhodanines and related compds. in screening campaigns, we decided to perform a systematic study on their promiscuity. An amt. of 163 rhodanines, hydantoins, thiohydantoins, and thiazolidinediones were synthesized and tested against several targets. The compds. were also characterized with respect to aggregation and electrophilic reactivity, and the binding modes of rhodanines and related compds. in published x-ray cocrystal structures were analyzed. The results indicate that the exocyclic, double bonded sulfur atom in rhodanines and thiohydantoins, in addn. to other structural features, offers a particularly high d. of interaction sites for polar interactions and hydrogen bonds. This causes a promiscuous behavior at concns. in the "screening range" but should not be regarded as a general knockout criterion that excludes such screening hits from further development. It is suggested that special criteria for target affinity and selectivity are applied to these classes of compds. and that their exceptional and potentially valuable biomol. binding properties are consequently exploited in a useful way.
- 195Schneider, T. H.; Rieger, M.; Ansorg, K.; Sobolev, A. N.; Schirmeister, T.; Engels, B.; Grabowsky, S. Vinyl Sulfone Building Blocks in Covalently Reversible Reactions with Thiols. New J. Chem. 2015, 39 (7), 5841– 5853, DOI: 10.1039/C5NJ00368G[Crossref], [CAS], Google Scholar195https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVWjt7jE&md5=2f65514bf92ec07ba1d344aa9b8a7599Vinyl sulfone building blocks in covalently reversible reactions with thiolsSchneider, Thomas H.; Rieger, Max; Ansorg, Kay; Sobolev, Alexandre N.; Schirmeister, Tanja; Engels, Bernd; Grabowsky, SimonNew Journal of Chemistry (2015), 39 (7), 5841-5853CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)In the present study we use quantum-chem. calcns. to investigate how the reactivity of vinyl sulfone-based compds. can be modified from an irreversible to a reversible reaction with thiols. Based on the predictions from theory, an array of nine different vinyl sulfones with systematically varying substitution pattern was synthesized and their crystal structures were detd. Subsequent Hirshfeld surface analyses employing the principle of electrostatic complementarity aid the understanding of the crystal packing of the synthesized compds. Reactivity studies against the nucleophile 2-phenylethanethiol mirror the properties predicted by the quantum-chem. computations in soln.
- 196Siklos, M.; BenAissa, M.; Thatcher, G. R. J. Cysteine Proteases as Therapeutic Targets: Does Selectivity Matter? A Systematic Review of Calpain and Cathepsin Inhibitors. Acta Pharm. Sin. B 2015, 5 (6), 506– 519, DOI: 10.1016/j.apsb.2015.08.001[Crossref], [PubMed], [CAS], Google Scholar196https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28rlslWjsg%253D%253D&md5=cbd246a39984c0d9e3a32fd26db8902dCysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitorsSiklos Marton; BenAissa Manel; Thatcher Gregory R JActa pharmaceutica Sinica. B (2015), 5 (6), 506-19 ISSN:2211-3835.Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.
- 197Cleary, J. A.; Doherty, W.; Evans, P.; Malthouse, J. P. G. Quantifying Tetrahedral Adduct Formation and Stabilization in the Cysteine and the Serine Proteases. Biochim. Biophys. Acta, Proteins Proteomics 2015, 1854, 1382– 1391, DOI: 10.1016/j.bbapap.2015.07.006[Crossref], [PubMed], [CAS], Google Scholar197https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFOktL%252FN&md5=f590715d70dc21d5553ba2c148a46796Quantifying tetrahedral adduct formation and stabilization in the cysteine and the serine proteasesCleary, Jennifer A.; Doherty, William; Evans, Paul; Malthouse, J. Paul G.Biochimica et Biophysica Acta, Proteins and Proteomics (2015), 1854 (10_Part_A), 1382-1391CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)Two new papain inhibitors have been synthesized where the terminal α-carboxyl groups of Z-Phe-Ala-COOH and Ac-Phe-Gly-COOH have been replaced by a proton to give Z-Phe-Ala-H and Ac-Phe-Gly-H. The authors show that for papain, replacing the terminal carboxylate group of a peptide inhibitor with a H atom decreased binding 3- to 4-fold, while replacing an aldehyde or glyoxal group with a H atom decreased binding by 300,000- to 1,000,000-fold. Thiohemiacetal formation by papain with aldehyde or glyoxal inhibitors was shown to be ∼10,000-fold more effective than hemiacetal or hemiketal formation with chymotrypsin. It was shown using effective molarities, that for papain, thiohemiacetal stabilization was more effective with aldehyde inhibitors than with glyoxal inhibitors. The effective molarity obtained when papain was inhibited by an aldehyde inhibitor was similar to the effective molarity obtained when chymotrypsin was inhibited by glyoxal inhibitors showing that both enzymes can stabilize tetrahedral adducts by similar amts. Therefore, the greater potency of aldehyde and glyoxal inhibitors with papain was not due to greater thiohemiacetal stabilization by papain compared to the hemiketal and hemiacetal stabilization by chymotrypsin, instead it reflected the greater intrinsic reactivity of the catalytic SH group of papain compared to the catalytic OH group of chymotrypsin. It was argued that while the hemiacetals and thiohemiacetals formed with the serine and cysteine proteases, resp., can mimic the catalytic tetrahedral intermediate they are also analogs of the productive and non-productive acyl intermediates that can be formed with the cysteine and serine proteases.
- 198Sanches, M.; Duffy, N. M.; Talukdar, M.; Thevakumaran, N.; Chiovitti, D.; Canny, M. D.; Lee, K.; Kurinov, I.; Uehling, D.; Al-awar, R.; Poda, G.; Prakesch, M.; Wilson, B.; Tam, V.; Schweitzer, C.; Toro, A.; Lucas, J. L.; Vuga, D.; Lehmann, L.; Durocher, D.; Zeng, Q.; Patterson, J. B.; Sicheri, F. Structure and Mechanism of Action of the Hydroxy–Aryl–Aldehyde Class of IRE1 Endoribonuclease Inhibitors. Nat. Commun. 2014, 5, 4202, DOI: 10.1038/ncomms5202[Crossref], [PubMed], [CAS], Google Scholar198https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksVertrk%253D&md5=bbdaaf0fad0ae104e3b1790bcc50df2bStructure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitorsSanches, Mario; Duffy, Nicole M.; Talukdar, Manisha; Thevakumaran, Nero; Chiovitti, David; Canny, Marella D.; Lee, Kenneth; Kurinov, Igor; Uehling, David; Al-Awar, Rima; Poda, Gennadiy; Prakesch, Michael; Wilson, Brian; Tam, Victor; Schweitzer, Colleen; Toro, Andras; Lucas, Julie L.; Vuga, Danka; Lehmann, Lynn; Durocher, Daniel; Zeng, Qingping; Patterson, John B.; Sicheri, FrankNature Communications (2014), 5 (), 4202CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Arom. ring systems contg. hydroxy-aldehyde moieties, termed hydroxy-aryl-aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chem. series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase-active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a hydrogen bond with Tyr892. Structure-activity studies and mutational anal. of contact residues define the optimal chem. space of inhibitors and validate the inhibitor-binding site. These studies lay the foundation for understanding both the biochem. and cellular functions of IRE1α using small mol. inhibitors and suggest new avenues for inhibitor design.
- 199Larraufie, M.-H.; Yang, W. S.; Jiang, E.; Thomas, A. G.; Slusher, B. S.; Stockwell, B. R. Incorporation of Metabolically Stable Ketones into a Small Molecule Probe to Increase Potency and Water Solubility. Bioorg. Med. Chem. Lett. 2015, 25 (21), 4787– 4792, DOI: 10.1016/j.bmcl.2015.07.018[Crossref], [PubMed], [CAS], Google Scholar199https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFOktL%252FF&md5=1816055cbe6493ea7336df8b59c18a35Incorporation of metabolically stable ketones into a small molecule probe to increase potency and water solubilityLarraufie, Marie-Helene; Yang, Wan Seok; Jiang, Elise; Thomas, Ajit G.; Slusher, Barbara S.; Stockwell, Brent R.Bioorganic & Medicinal Chemistry Letters (2015), 25 (21), 4787-4792CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Introducing a reactive carbonyl to a scaffold that does not otherwise have an electrophilic functionality to create a reversible covalent inhibitor is a potentially useful strategy for enhancing compd. potency. However, aldehydes are metabolically unstable, which precludes the use of this strategy for compds. to be tested in animal models or in human clin. studies. To overcome this limitation, the authors designed ketone-based functionalities capable of forming reversible covalent adducts, while displaying high metabolic stability, and imparting improved water soly. to their pendant scaffold. The authors tested this strategy on the ferroptosis inducer and exptl. therapeutic erastin, and obsd. substantial increases in compd. potency. In particular, a new carbonyl erastin analog, termed IKE, displayed improved potency, soly. and metabolic stability, thus representing an ideal candidate for future in vivo cancer therapeutic applications.
- 200Cross, B. C. S.; Bond, P. J.; Sadowski, P. G.; Jha, B. K.; Zak, J.; Goodman, J. M.; Silverman, R. H.; Neubert, T. A.; Baxendale, I. R.; Ron, D.; Harding, H. P. The Molecular Basis for Selective Inhibition of Unconventional MRNA Splicing by an IRE1-Binding Small Molecule. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (15), E869– E878, DOI: 10.1073/pnas.1115623109[Crossref], [PubMed], [CAS], Google Scholar200https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmtVCgtb8%253D&md5=f6a05686d1b4a8a25d33fea41270f3eeThe molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small moleculeCross, Benedict C. S.; Bond, Peter J.; Sadowski, Pawel G.; Jha, Babal Kant; Zak, Jaroslav; Goodman, Jonathan M.; Silverman, Robert H.; Neubert, Thomas A.; Baxendale, Ian R.; Ron, David; Harding, Heather P.Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (15), E869-E878, SE869/1-SE869/13CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degrdn. of diverse membrane-bound mRNAs. We report on the identification of a small mol. inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degrdn. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chem. reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathol. settings.
- 201Knoepfel, T.; Furet, P.; Mah, R.; Buschmann, N.; Leblanc, C.; Ripoche, S.; Graus-Porta, D.; Wartmann, M.; Galuba, I.; Fairhurst, R. A. 2-Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4. ACS Med. Chem. Lett. 2018, 9 (3), 215– 220, DOI: 10.1021/acsmedchemlett.7b00485[ACS Full Text], [CAS], Google Scholar201https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit1Srsb4%253D&md5=fd43811b8a9e5f7a02f27096569423f82-Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4Knoepfel, Thomas; Furet, Pascal; Mah, Robert; Buschmann, Nicole; Leblanc, Catherine; Ripoche, Sebastien; Graus-Porta, Diana; Wartmann, Markus; Galuba, Inga; Fairhurst, Robin A.ACS Medicinal Chemistry Letters (2018), 9 (3), 215-220CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)As part of a project to identify FGFR4 selective inhibitors, scaffold morphing of a 2-formylquinoline amide hit identified series of 2-formylpyridine ureas (2-FPUs) with improved potency and physicochem. properties. In particular, tetrahydronaphthyridine urea analogs with cellular activities below 30 nM have been identified. Consistent with the hypothesized reversible-covalent mechanism of inhibition, the 2-FPUs exhibited slow binding kinetics, and the aldehyde, as the putative electrophile, could be demonstrated to be a key structural element for activity.
- 202LoPachin, R. M.; Gavin, T. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective. Chem. Res. Toxicol. 2014, 27 (7), 1081– 1091, DOI: 10.1021/tx5001046[ACS Full Text], [CAS], Google Scholar202https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpsVensrg%253D&md5=e72707796ea59ccaff0283848ebbdf76Molecular Mechanisms of Aldehyde Toxicity: A Chemical PerspectiveLoPachin, Richard M.; Gavin, TerrenceChemical Research in Toxicology (2014), 27 (7), 1081-1091CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)A review. Aldehydes are electrophilic compds. to which humans are pervasively exposed. Despite a significant health risk due to exposure, the mechanisms of aldehyde toxicity are poorly understood. This ambiguity is likely due to the structural diversity of aldehyde derivs. and corresponding differences in chem. reactions and biol. targets. To gain mechanistic insight, the authors have used parameters based on the hard and soft, acids and bases (HSAB) theory to profile the different aldehyde subclasses with respect to electronic character (softness, hardness), electrophilic reactivity (electrophilic index), and biol. nucleophilic targets. The authors' analyses indicate that short chain aldehydes and longer chain satd. alkanals are hard electrophiles that cause toxicity by forming adducts with hard biol. nucleophiles, e.g., primary nitrogen groups on lysine residues. In contrast, α,β-unsatd. carbonyl derivs., alkenals, and the α-oxoaldehydes are soft electrophiles that preferentially react with soft nucleophilic thiolate groups on cysteine residues. The aldehydes can therefore be grouped into subclasses according to common electronic characteristics (softness/hardness) and mol. mechanisms of toxicity. As the authors will discuss, the toxic potencies of these subgroups are generally related to corresponding electrophilicities. For some aldehydes, however, predictions of toxicity based on electrophilicity are less accurate due to inherent physicochem. variables that limit target accessibility, e.g., steric hindrance and soly. The unsatd. aldehydes are also members of the conjugated type-2 alkene chem. class that includes α,β-unsatd. amide, ketone, and ester derivs. Type-2 alkenes are electrophiles of varying softness and electrophilicity that share a common mechanism of toxicity. Therefore, exposure to an environmental mixt. of unsatd. carbonyl derivs. could cause "type-2 alkene toxicity" through additive interactions. Finally, the authors propose that environmentally derived aldehydes can accelerate diseases by interacting with endogenous aldehydes generated during oxidative stress. This review provides a basis for understanding aldehyde mechanisms and environmental toxicity through the context of electronic structure, electrophilicity, and nucleophile target selectivity.
- 203Caraballo, R.; Dong, H.; Ribeiro, J. P.; Jiménez-Barbero, J.; Ramström, O. Direct STD NMR Identification of β-Galactosidase Inhibitors from a Virtual Dynamic Hemithioacetal System. Angew. Chem., Int. Ed. 2010, 49 (3), 589– 593, DOI: 10.1002/anie.200903920[Crossref], [CAS], Google Scholar203https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktlaksw%253D%253D&md5=012f6210d6e743fe5dd958cc4500d937Direct STD NMR Identification of β-Galactosidase Inhibitors from a Virtual Dynamic Hemithioacetal SystemCaraballo, Remi; Dong, Hai; Ribeiro, Joao P.; Jimenez-Barbero, Jesus; Ramstroem, OlofAngewandte Chemie, International Edition (2010), 49 (3), 589-593, S589/1-S589/20CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Herein we show that hemithioacetal (HTA) formation is a fast and efficient reversible reaction that can be used to generate systems of potential enzyme inhibitors in aq. media under neutral conditions. These systems were also subjected to direct binding anal. by satn. transfer difference (STD) NMR spectroscopy, a rapid and efficient technique for protein-ligand binding studies, in which the optimal HTA interactions with the biol. target can be readily deduced. We have successfully demonstrated, for the first time, hemithioacetal formation applied to dynamic combinatorial system generation in aq. media. Equil. formation using this chem. proved very rapid and resulted in truly virtual dynamic systems in which 1H NMR analyses confirmed the presence of transient HTA constituents. It has further been demonstrated that 1H STD NMR spectroscopy can be used for efficient and direct in situ identification of the best enzyme binders of the virtual dynamic system. Inhibition studies unequivocally support the 1H STD NMR.exptl. data and enable the distinction of different β-galactosidase inhibitors and nonspecific binders.
- 204Buschmann, N.; Fairhurst, R. A.; Knoepfel, T.; Furet, P.; Leblanc, C.; Mah, R.; Kiffe, M.; Graus-Porta, D.; Weiss, A.; Kinyamu-Akunda, J.; Wartmann, M.; Trappe, J.; Gabriel, T. R.; Hofmann, F.; Sellers, W. R. A Reversible Covalent Approach to Selective FGFR4 Inhibition; Book of Abstracts—Frontiers in Medicinal Chemistry Symposium, Jena, 2018; p 26.
- 205Otto, H.-H.; Schirmeister, T. Cysteine Proteases and Their Inhibitors. Chem. Rev. 1997, 97 (1), 133– 172, DOI: 10.1021/cr950025u[ACS Full Text], [CAS], Google Scholar205https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXktFOhsA%253D%253D&md5=0c38c0f09f14ad664b6ca707ce08a5b9Cysteine Proteases and Their InhibitorsOtto, Hans-Hartwig; Schirmeister, TanjaChemical Reviews (Washington, D. C.) (1997), 97 (1), 133-171CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with 599 refs. Some typical examples of cysteine proteases from different sources are characterized in terms of property, structure and specificity. Possible functions of human and mammalian cysteine proteases, lysosomal cathepsins and cytoplasmic calpains are discussed, as well as their role in pathol. processes. In addn. to the endogenous inhibitors, the cystatins and calpastatins, the currently available synthetic inhibitors are also described together with their structures, reaction mechanisms, activities and selectivities. Potential medical applications of cysteine protease inhibitors are discussed. Particular emphasis is given to comparison of cysteine proteases with serine proteases, due to their similar proteolytic mechanisms.
- 206Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.; Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.; Huang, Q.; Han, S.-P.; Abboa-Offei, B.; Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.; Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. Discovery and Preclinical Profile of Saxagliptin (BMS-477118): A Highly Potent, Long-Acting, Orally Active Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 Diabetes. J. Med. Chem. 2005, 48 (15), 5025– 5037, DOI: 10.1021/jm050261p[ACS Full Text], [CAS], Google Scholar206https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXlsVeltb0%253D&md5=878355880133bb0ddd6bd14ecbf1d070Discovery and Preclinical Profile of Saxagliptin (BMS-477118): A Highly Potent, Long-Acting, Orally Active Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 DiabetesAugeri, David J.; Robl, Jeffrey A.; Betebenner, David A.; Magnin, David R.; Khanna, Ashish; Robertson, James G.; Wang, Aiying; Simpkins, Ligaya M.; Taunk, Prakash; Huang, Qi; Han, Song-Ping; Abboa-Offei, Benoni; Cap, Michael; Xin, Li; Tao, Li; Tozzo, Effie; Welzel, Gustav E.; Egan, Donald M.; Marcinkeviciene, Jovita; Chang, Shu Y.; Biller, Scott A.; Kirby, Mark S.; Parker, Rex A.; Hamann, Lawrence G.Journal of Medicinal Chemistry (2005), 48 (15), 5025-5037CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Efforts to further elucidate structure-activity relationships (SAR) within the authors previously disclosed series of β-quaternary amino acid linked L-cis-4,5-methanoprolinenitrile dipeptidyl peptidase IV (DPP-IV) inhibitors led to the investigation of vinyl substitution at the β-position of α-cycloalkyl-substituted glycines. Despite poor systemic exposure, vinyl-substituted compds. showed extended duration of action in acute rat ex vivo plasma DPP-IV inhibition models. Oxygenated putative metabolites were prepd. and were shown to exhibit the potency and extended duration of action of their precursors in efficacy models measuring glucose clearance in Zuckerfa/fa rats. Extension of this approach to adamantylglycine-derived inhibitors led to the discovery of highly potent inhibitors, including hydroxyadamantyl compd. BMS-477118 (saxagliptin), a highly efficacious, stable, and long-acting DPP-IV inhibitor, which is currently undergoing clin. trials for treatment of type 2 diabetes.
- 207Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C. Nitrile-Containing Pharmaceuticals: Efficacious Roles of the Nitrile Pharmacophore. J. Med. Chem. 2010, 53 (22), 7902– 7917, DOI: 10.1021/jm100762r[ACS Full Text], [CAS], Google Scholar207https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtV2kurvP&md5=040bbaebd9e8c9999d195b9a22221feaNitrile-Containing Pharmaceuticals: Efficacious Roles of the Nitrile PharmacophoreFleming, Fraser F.; Yao, Lihua; Ravikumar, P. C.; Funk, Lee; Shook, Brian C.Journal of Medicinal Chemistry (2010), 53 (22), 7902-7917CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. The review is structured according to the nature of the nitrile-bearing substituent. As the no. of nitrile-contg. drug leads is vast, the review has focused on launched nitrile-contg. pharmaceuticals and currently active clin. candidates. Most nitrile-contg. pharmaceuticals are aroms. with aliph.-, alkene-, and nitrogen-bearing nitriles being progressively less frequent. Within each class, the bioactive nitriles are collated according to common structural elements and mode of action. The hope is that this survey will allow greater deployment of this versatile functionality within drug leads.
- 208Oballa, R. M.; Truchon, J.-F.; Bayly, C. I.; Chauret, N.; Day, S.; Crane, S.; Berthelette, C. A Generally Applicable Method for Assessing the Electrophilicity and Reactivity of Diverse Nitrile-Containing Compounds. Bioorg. Med. Chem. Lett. 2007, 17 (4), 998– 1002, DOI: 10.1016/j.bmcl.2006.11.044[Crossref], [PubMed], [CAS], Google Scholar208https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht12qtr4%253D&md5=b8fff00bdd6a746753f8f244d25b0cc8A generally applicable method for assessing the electrophilicity and reactivity of diverse nitrile-containing compoundsOballa, Renata M.; Truchon, Jean-Francois; Bayly, Christopher I.; Chauret, Nathalie; Day, Stephen; Crane, Sheldon; Berthelette, CarlBioorganic & Medicinal Chemistry Letters (2007), 17 (4), 998-1002CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)Nitrile-based inhibitors of cathepsin K have been known for some time and mechanism-of-action studies have demonstrated that cysteinyl proteases interact with nitriles in a reversible fashion. Three main classes of nitrile-contg. inhibitors have been published in the cathepsin K field: (i) cyanamides, (ii) arom. nitriles, and (iii) aminoacetonitriles. A computational approach was used to calc. the theor. reactivities of diverse nitriles and this was found to correlate with their extent of reactivity with free cysteine. Moreover, there is a tentative link between high reactivity with cysteine and the potential to lead to irreversible covalent binding to proteins.
- 209Schmitz, J.; Beckmann, A.-M.; Dudic, A.; Li, T.; Sellier, R.; Bartz, U.; Gütschow, M. 3-Cyano-3-Aza-β-Amino Acid Derivatives as Inhibitors of Human Cysteine Cathepsins. ACS Med. Chem. Lett. 2014, 5 (10), 1076– 1081, DOI: 10.1021/ml500238q[ACS Full Text], [CAS], Google Scholar209https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlalu7rL&md5=2c500ebf40c879cfbd905fd57ba006863-Cyano-3-aza-β-amino Acid Derivatives as Inhibitors of Human Cysteine CathepsinsSchmitz, Janina; Beckmann, Anna-Madeleine; Dudic, Adela; Li, Tianwei; Sellier, Robert; Bartz, Ulrike; Guetschow, MichaelACS Medicinal Chemistry Letters (2014), 5 (10), 1076-1081CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Nitrile-type inhibitors are known to interact with cysteine proteases in a covalent-reversible manner. The chemotype of 3-cyano-3-aza-β-amino acid derivs. I [R1 = H, Me; R2 = Me, Bn; R3 = Bn; R2R3 = 4-morpholinyl] was designed in which the N-cyano group is centrally arranged in the mol. to allow for interactions with the nonprimed and primed binding regions of the target enzymes. These compds. were evaluated as inhibitors of the human cysteine cathepsins K, S, B, and L. They exhibited slow-binding behavior and were found to be exceptionally potent, in particular toward cathepsin K, with second-order rate consts. up to 52 900 × 103 M-1s-1.
- 210Cai, J.; Fradera, X.; van Zeeland, M.; Dempster, M.; Cameron, K. S.; Bennett, D. J.; Robinson, J.; Popplestone, L.; Baugh, M.; Westwood, P.; Bruin, J.; Hamilton, W.; Kinghorn, E.; Long, C.; Uitdehaag, J. C. M. 4-(3-Trifluoromethylphenyl)-Pyrimidine-2-Carbonitrile as Cathepsin S Inhibitors: N3, Not N1 Is Critically Important. Bioorg. Med. Chem. Lett. 2010, 20 (15), 4507– 4510, DOI: 10.1016/j.bmcl.2010.06.043[Crossref], [PubMed], [CAS], Google Scholar210https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXptVSktL8%253D&md5=c4f3a722e29616dca5e47b0c8cbcadef4-(3-Trifluoromethylphenyl)-pyrimidine-2-carbonitrile as cathepsin S inhibitors: N3, not N1 is critically importantCai, Jiaqiang; Fradera, Xavier; van Zeeland, Mario; Dempster, Maureen; Cameron, Kenneth S.; Bennett, D. Jonathan; Robinson, John; Popplestone, Lucy; Baugh, Mark; Westwood, Paul; Bruin, John; Hamilton, William; Kinghorn, Emma; Long, Clive; Uitdehaag, Joost C. M.Bioorganic & Medicinal Chemistry Letters (2010), 20 (15), 4507-4510CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Using computer aided modeling studies, a new extended P2/S2 interaction was identified. This extended region can accommodate a variety of functional groups, such as aryls and basic amines. It was discovered that the N3 nitrogen of the pyrimidine-2-carbonitrile is crit. for its cathepsin cysteine protease inhibition. N1 nitrogen also contributes to the inhibitory activity, but to a very limited degree. An in situ double activation' mechanism was proposed to explain these results.
- 211Mac Sweeney, A.; Grosche, P.; Ellis, D.; Combrink, K.; Erbel, P.; Hughes, N.; Sirockin, F.; Melkko, S.; Bernardi, A.; Ramage, P.; Jarousse, N.; Altmann, E. Discovery and Structure-Based Optimization of Adenain Inhibitors. ACS Med. Chem. Lett. 2014, 5 (8), 937– 941, DOI: 10.1021/ml500224t[ACS Full Text], [CAS], Google Scholar211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M%252FktFWmsg%253D%253D&md5=d3de233ac94209da27bcef375970a33cDiscovery and structure-based optimization of adenain inhibitorsMac Sweeney Aengus; Grosche Philipp; Erbel Paul; Hughes Nicola; Sirockin Finton; Melkko Samu; Bernardi Anna; Ramage Paul; Altmann Eva; Ellis David; Combrink Keith; Jarousse NadineACS medicinal chemistry letters (2014), 5 (8), 937-41 ISSN:1948-5875.The cysteine protease adenain is the essential protease of adenovirus and, as such, represents a promising target for the treatment of ocular and other adenoviral infections. Through a concise two-pronged hit discovery approach we identified tetrapeptide nitrile 1 and pyrimidine nitrile 2 as complementary starting points for adenain inhibition. These hits enabled the first high-resolution X-ray cocrystal structures of adenain with inhibitors bound and revealed the binding mode of 1 and 2. The screening hits were optimized by a structure-guided medicinal chemistry strategy into low nanomolar drug-like inhibitors of adenain.
- 212de Jesus Cortez, F.; Nguyen, P.; Truillet, C.; Tian, B.; Kuchenbecker, K. M.; Evans, M. J.; Webb, P.; Jacobson, M. P.; Fletterick, R. J.; England, P. M. Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogen. ACS Chem. Biol. 2017, 12 (12), 2934– 2939, DOI: 10.1021/acschembio.7b00702[ACS Full Text], [CAS], Google Scholar212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1Sksr%252FP&md5=3871dfbf0cd53790a8e63f84a1157416Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogende Jesus Cortez, Felipe; Nguyen, Phuong; Truillet, Charles; Tian, Boxue; Kuchenbecker, Kristopher M.; Evans, Michael J.; Webb, Paul; Jacobson, Matthew P.; Fletterick, Robert J.; England, Pamela M.ACS Chemical Biology (2017), 12 (12), 2934-2939CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Resistance to clin. antiandrogens has plagued the evolution of effective therapeutics for advanced prostate cancer. As with the first-line therapeutic bicalutamide (Casodex), resistance to newer antiandrogens (enzalutamide, ARN-509) develops quickly in patients, despite the fact that these drugs have ∼10-fold better affinity for androgen receptor than bicalutamide. Improving affinity alone is often not sufficient to prevent resistance and alternative strategies are needed to improve antiandrogen efficacy. Covalent and reversible covalent drugs are being used to thwart drug resistance in other contexts, and activated aryl nitriles are among the moieties being exploited for this purpose. The authors capitalized on the presence of an aryl nitrile in bicalutamide, and the existence of a native cysteine residue (Cys784) in the androgen receptor ligand binding pocket, to develop 5N-bicalutamide I, a cysteine-reactive antiandrogen. I exhibits a 150-fold improvement in Ki and 20-fold improvement in IC50 over the parent compd. The authors attribute the marked improvement in affinity and activity to the formation of a covalent adduct with Cys784, a residue that is not among the more than 160 androgen receptor point mutations assocd. with prostate cancer. Increasing the residence time of bound antiandrogen via formation of a covalent adduct may forestall the drug resistance seen with current clin. antiandrogens.
- 213Deaton, D. N.; Hassell, A. M.; McFadyen, R. B.; Miller, A. B.; Miller, L. R.; Shewchuk, L. M.; Tavares, F. X.; Willard, D. H.; Wright, L. L. Novel and Potent Cyclic Cyanamide-Based Cathepsin K Inhibitors. Bioorg. Med. Chem. Lett. 2005, 15 (7), 1815– 1819, DOI: 10.1016/j.bmcl.2005.02.033[Crossref], [PubMed], [CAS], Google Scholar213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisVCntL8%253D&md5=7d41d58899ce26c59a839a041149cd53Novel and potent cyclic cyanamide-based cathepsin K inhibitorsDeaton, David N.; Hassell, Anne M.; McFadyen, Robert B.; Miller, Aaron B.; Miller, Larry R.; Shewchuk, Lisa M.; Tavares, Francis X.; Willard, Derril H.; Wright, Lois L.Bioorganic & Medicinal Chemistry Letters (2005), 15 (7), 1815-1819CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Starting from a PDE IV inhibitor hit derived from high throughput screening of the compd. collection, a key pyrrolidine cyanamide pharmacophore was identified. Modifications of the pyrrolidine ring produced enhancements in cathepsin K inhibition. An X-ray co-crystal structure of a cyanamide with cathepsin K confirmed the mode of inhibition.
- 214Falgueyret, J.-P.; Oballa, R. M.; Okamoto, O.; Wesolowski, G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.; Riendeau, D.; Rodan, S. B.; Percival, M. D. Novel, Nonpeptidic Cyanamides as Potent and Reversible Inhibitors of Human Cathepsins K and L. J. Med. Chem. 2001, 44 (1), 94– 104, DOI: 10.1021/jm0003440[ACS Full Text], [CAS], Google Scholar214https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXos1ajs7g%253D&md5=aed27c1353797e413890ea02d900095bNovel, Nonpeptidic Cyanamides as Potent and Reversible Inhibitors of Human Cathepsins K and LFalgueyret, Jean-Pierre; Oballa, Renata M.; Okamoto, Osamu; Wesolowski, Gregg; Aubin, Yves; Rydzewski, Robert M.; Prasit, Peppi; Riendeau, Denis; Rodan, Sevgi B.; Percival, M. DavidJournal of Medicinal Chemistry (2001), 44 (1), 94-104CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Compds. contg. a 1-cyanopyrrolidinyl ring were identified as potent and reversible inhibitors of cathepsins K and L. The original lead compd. I inhibits cathepsins K and L with IC50 values of 0.37 and 0.45 μM, resp. Modification of compd. I by replacement of the quinoline moiety led to the synthesis of N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide (2). Compd. II was found to be a potent inhibitor of cathepsins K and L with a Ki value of 50 nM for cathepsin K. Replacement of the 1-cyanopyrrolidine of compd. II by a 1-cyanoazetidine increased the potency of the inhibitor by 10-fold. This increase in potency is probably due to an enhanced chem. reactivity of the compd. toward the thiolate of the active site of the enzyme. This is demonstrated when the assay is performed in the presence of glutathione at pH 7.0 which favors the formation of a GSH thiolate anion. Under these assay conditions, there is a loss of potency in the 1-cyanoazetidine series due to the formation of an inactive complex between the GSH thiolate and the 1-cyanoazetidine inhibitors. 1-Cyanopyrrolidinyl inhibitors exhibited time-dependent inhibition which allowed us to det. the assocn. and dissocn. rate consts. with human cathepsin K. The kinetic data obtained showed that the increase of potency obsd. between different 1-cyanopyrrolidinyl inhibitors is due to an increase of kon values and that the assocn. of the compd. with the enzyme fits an apparent one-step mechanism. 13C NMR expts. performed with the enzyme papain showed that compd. 2 forms a covalent isothiourea ester adduct with the enzyme. As predicted by the kinetic anal., the addn. of the irreversible inhibitor E64 to the enzyme-cyanopyrrolidinyl complex totally abolished the signal of the isothiourea bond as obsd. by 13C NMR, thereby demonstrating that the formation of the covalent bond with the active site cysteine residue is reversible. Finally, compd. II inhibits bone resorption in an in vitro assay involving rabbit osteoclasts and bovine bone with an IC50 value of 0.7 μM. 1-Cyanopyrrolidine represents a new class of nonpeptidic compds. that inhibit cathepsin K and L activity and proteolysis of bone collagen.
- 215Rydzewski, R. M.; Bryant, C.; Oballa, R.; Wesolowski, G.; Rodan, S. B.; Bass, K. E.; Wong, D. H. Peptidic 1-Cyanopyrrolidines: Synthesis and SAR of a Series of Potent, Selective Cathepsin Inhibitors. Bioorg. Med. Chem. 2002, 10 (10), 3277– 3284, DOI: 10.1016/S0968-0896(02)00173-6[Crossref], [PubMed], [CAS], Google Scholar215https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XlvVems7o%253D&md5=4e40001c5fe70cbf3cb309bb8333bb5dPeptidic 1-cyanopyrrolidines: synthesis and SAR of a series of potent, selective cathepsin inhibitorsRydzewski, Robert M.; Bryant, Clifford; Oballa, Renata; Wesolowski, Gregg; Rodan, Sevgi B.; Bass, Kathryn E.; Wong, Darren H.Bioorganic & Medicinal Chemistry (2002), 10 (10), 3277-3284CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)1-Cyanopyrrolidines have previously been reported to inhibit cysteinyl cathepsins (Falgueyret, J.-P. et al., J. Med. Chem. 2001, 44, 94). To optimize binding interactions for a given cathepsin and simultaneously reduce interactions with the other closely related enzymes, small peptidic substituents were introduced to the 1-cyanopyrrolidine scaffold, either at the 2-position starting with proline or at the 3-position of aminopyrrolidines. The resulting novel compds. proved to be micromolar inhibitors of cathepsin B (Cat B) but nanomolar to picomolar inhibitors of cathepsins K, L, and S (Cat K, Cat L, Cat S). Several of the compds. were >20-fold selective vs. the other 3 cathepsins. SAR trends were obsd., most notably the remarkable potency of Cat L inhibitors based on the 1-cyano-d-proline scaffold. The selectivity of one such compd., the 94 picomolar Cat L inhibitor (I), was demonstrated at higher concns. in DLD-1 cells. Although none of the compds. in the proline series that was tested proved to be submicromolar in the in vitro bone resorption assay, 2 Cat K inhibitors in the 3-substituted pyrrolidine series were relatively potent in that assay.
- 216Lainé, D.; Palovich, M.; McCleland, B.; Petitjean, E.; Delhom, I.; Xie, H.; Deng, J.; Lin, G.; Davis, R.; Jolit, A.; Nevins, N.; Zhao, B.; Villa, J.; Schneck, J.; McDevitt, P.; Midgett, R.; Kmett, C.; Umbrecht, S.; Peck, B.; Davis, A. B.; Bettoun, D. Discovery of Novel Cyanamide-Based Inhibitors of Cathepsin C. ACS Med. Chem. Lett. 2011, 2 (2), 142– 147, DOI: 10.1021/ml100212k[ACS Full Text], [CAS], Google Scholar216https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtl2rsL%252FK&md5=5b8478f6abc28e4d273eb9aa7c93aeb1Discovery of novel cyanamide-based inhibitors of cathepsin CLaine, Dramane; Palovich, Michael; McCleland, Brent; Petitjean, Emilie; Delhom, Isabelle; Xie, Haibo; Deng, Jianghe; Lin, Guoliang; Davis, Roderick; Jolit, Anais; Nevins, Neysa; Zhao, Baoguang; Villa, Jim; Schneck, Jessica; McDevitt, Patrick; Midgett, Robert; Kmett, Casey; Umbrecht, Sandra; Peck, Brian; Davis, Alicia Bacon; Bettoun, DavidACS Medicinal Chemistry Letters (2011), 2 (2), 142-147CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)The discovery of potent and selective cyanamide-based inhibitors of the cysteine protease cathepsin C is detailed. Optimization of the template with regard to plasma stability led to the identification of (2S,4R)-1-cyano-2-methyl-4-(2,5-dibromophenylsulfonylamino)pyrrolidine (I), a potent cathepsin C inhibitor with excellent selectivity over other cathepsins and potent in vivo activity in a cigarette smoke mouse model.
- 217Hill, S. V.; Williams, A.; Longridge, J. L. Acid-Catalysed Hydrolysis of Cyanamides: Estimates of Carbodi-Imide Basicity and Tautomeric Equilibrium Constant between Carbodi-Imide and Cyanamide. J. Chem. Soc., Perkin Trans. 2 1984, 0 (6), 1009– 1013, DOI: 10.1039/p29840001009[Crossref], [CAS], Google Scholar217https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXlslSgsL8%253D&md5=e142ad1a3c0bca71daf9eec01035e538Acid-catalyzed hydrolysis of cyanamides: estimates of carbodiimide basicity and tautomeric equilibrium constant between carbodiimide and cyanamideHill, Stephen V.; Williams, Andrew; Longridge, Jethro L.Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1984), (6), 1009-13CODEN: JCPKBH; ISSN:0300-9580.N-Cyanourea is an intermediate in the hydrolysis of NCNHCN (I), which is shown to react as its carbodiimide tautomer. The hydrolysis of I, H2NCONHCN, and (MeNH)2CNCN (II) is specific acid-catalyzed. General acid catalysis is demonstrated for the hydrolysis of II which is considered to be specific acid-nucleophilic. Assuming the carbodiimide mechanism also holds for the specific acid-catalyzed hydrolysis of H2NCN, the tautomeric equil. const. for formation of the parent carbodiimide in H2O at 25° is 0.6 × 10-7 and the pK of the protonated carbodiimide is estd. to be -1 to +1. Rate consts. are reported for alk. hydrolysis of cyanamides.
- 218Benson, M. J.; Rodriguez, V.; von Schack, D.; Keegan, S.; Cook, T. A.; Edmonds, J.; Benoit, S.; Seth, N.; Du, S.; Messing, D.; Nickerson-Nutter, C. L.; Dunussi-Joannopoulos, K.; Rankin, A. L.; Ruzek, M.; Schnute, M. E.; Douhan, J. Modeling the Clinical Phenotype of BTK Inhibition in the Mature Murine Immune System. J. Immunol. 2014, 193 (1), 185, DOI: 10.4049/jimmunol.1302570[Crossref], [PubMed], [CAS], Google Scholar218https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVWmsLbJ&md5=817796f74f02afc4171dbb6b19368528Modeling the Clinical Phenotype of BTK Inhibition in the Mature Murine Immune SystemBenson, Micah J.; Rodriguez, Varenka; von Schack, David; Keegan, Sean; Cook, Tim A.; Edmonds, Jason; Benoit, Stephen; Seth, Nilufer; Du, Sarah; Messing, Dean; Nickerson-Nutter, Cheryl L.; Dunussi-Joannopoulos, Kyri; Rankin, Andrew L.; Ruzek, Melanie; Schnute, Mark E.; Douhan, JohnJournal of Immunology (2014), 193 (1), 185-197CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncol. and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clin. phenotype of BTK inhibition by systematically examg. the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clin. phenotype of chronic BTK inhibition.
- 219Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.; Almaden, C.; Nagata, A.; Ryan, K.; Feng, J.; Dalvie, D.; Kath, J. C.; Xu, M.; Wani, R.; Murray, B. W. Covalent EGFR Inhibitor Analysis Reveals Importance of Reversible Interactions to Potency and Mechanisms of Drug Resistance. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (1), 173– 178, DOI: 10.1073/pnas.1313733111[Crossref], [PubMed], [CAS], Google Scholar219https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXps1eluw%253D%253D&md5=47197c5e7876a238eefb2e8e7de305eaCovalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistanceSchwartz, Phillip A.; Kuzmic, Petr; Solowiej, James; Bergqvist, Simon; Bolanos, Ben; Almaden, Chau; Nagata, Asako; Ryan, Kevin; Feng, Junli; Dalvie, Deepak; Kath, John C.; Xu, Meirong; Wani, Revati; Murray, Brion WilliamProceedings of the National Academy of Sciences of the United States of America (2014), 111 (1), 173-178CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chem. reactivity in overall potency are not well-understood. To characterize the underlying mol. processes at a microscopic level and det. the appropriate kinetic consts., specialized exptl. design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 105-107 M-1s-1), despite their low specific reactivity (kinact ≤ 2.1 × 10-3 s-1), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissocn. and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidn. on enzyme catalysis and inhibitor pharmacol. are characterized. Oxidn. of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidn. (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors.
- 220Zapf, C. W.; Gerstenberger, B. S.; Xing, L.; Limburg, D. C.; Anderson, D. R.; Caspers, N.; Han, S.; Aulabaugh, A.; Kurumbail, R.; Shakya, S.; Li, X.; Spaulding, V.; Czerwinski, R. M.; Seth, N.; Medley, Q. G. Covalent Inhibitors of Interleukin-2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood Assay. J. Med. Chem. 2012, 55 (22), 10047– 10063, DOI: 10.1021/jm301190s[ACS Full Text], [CAS], Google Scholar220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFOlsbnP&md5=7e39a17151dc9493d13439bc48fa83a3Covalent Inhibitors of Interleukin-2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood AssayZapf, Christoph W.; Gerstenberger, Brian S.; Xing, Li; Limburg, David C.; Anderson, David R.; Caspers, Nicole; Han, Seungil; Aulabaugh, Ann; Kurumbail, Ravi; Shakya, Subarna; Li, Xin; Spaulding, Vikki; Czerwinski, Robert M.; Seth, Nilufer; Medley, Quintus G.Journal of Medicinal Chemistry (2012), 55 (22), 10047-10063CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concns. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogs are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approx. 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.
- 221Gupta, P.; Wright, S. E.; Kim, S.-H.; Srivastava, S. K. Phenethyl Isothiocyanate: A Comprehensive Review of Anti-Cancer Mechanisms. Biochim. Biophys. Acta, Rev. Cancer 2014, 1846 (2), 405– 424, DOI: 10.1016/j.bbcan.2014.08.003[Crossref], [CAS], Google Scholar221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVKnt7fM&md5=ba0ae7ed36d1f72329d7257393f24df7Phenethyl isothiocyanate: A comprehensive review of anti-cancer mechanismsGupta, Parul; Wright, Stephen E.; Kim, Sung-Hoon; Srivastava, Sanjay K.Biochimica et Biophysica Acta, Reviews on Cancer (2014), 1846 (2), 405-424CODEN: BBACEU; ISSN:0304-419X. (Elsevier B.V.)A review. The epidemiol. evidence suggests a strong inverse relationship between dietary intake of cruciferous vegetables and the incidence of cancer. Among other constituents of cruciferous vegetables, isothiocyanates (ITC) are the main bioactive chems. present. Phenethyl isothiocyanate (PEITC) is present as gluconasturtiin in many cruciferous vegetables with remarkable anti-cancer effects. PEITC is known to not only prevent the initiation phase of carcinogenesis process but also to inhibit the progression of tumorigenesis. PEITC targets multiple proteins to suppress various cancer-promoting mechanisms such as cell proliferation, progression and metastasis. Pre-clin. evidence suggests that combination of PEITC with conventional anti-cancer agents is also highly effective in improving overall efficacy. Based on accumulating evidence, PEITC appears to be a promising agent for cancer therapy and is already under clin. trials for leukemia and lung cancer. This is the first review which provides a comprehensive anal. of known targets and mechanisms along with a crit. evaluation of PEITC as a future anti-cancer agent.
- 222Hinman, A.; Chuang, H.; Bautista, D. M.; Julius, D. TRP Channel Activation by Reversible Covalent Modification. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (51), 19564– 19568, DOI: 10.1073/pnas.0609598103[Crossref], [PubMed], [CAS], Google Scholar222https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhsVGksg%253D%253D&md5=3fb76ecf2a1d5d0992ade1873b2d5bccTRP channel activation by reversible covalent modificationHinman, Andrew; Chuang, Huai-hu; Bautista, Diana M.; Julius, DavidProceedings of the National Academy of Sciences of the United States of America (2006), 103 (51), 19564-19568CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Allyl isothiocyanate, the pungent principle of wasabi and other mustard oils, produces pain by activating TRPA1, an excitatory ion channel on sensory nerve endings. Isothiocyanates are membrane--permeable electrophiles that form adducts with thiols and primary amines, suggesting that covalent modification, rather than classical lock-and-key binding, accounts for their agonist properties. Indeed, we show that the thio1 reactive compds. of diverse structure activate TRPA1 in a manner that relies on covalent modification of cysteine residues within the cytoplasmic N terminus of the channel. These findings suggest an unusual paradigm whereby natural products activate a receptor through direct, reversible, and covalent protein modification.
- 223van Bladeren, P. J. Glutathione Conjugation as a Bioactivation Reaction. Chem.-Biol. Interact. 2000, 129 (1), 61– 76, DOI: 10.1016/S0009-2797(00)00214-3[Crossref], [PubMed], [CAS], Google Scholar223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXitlOntA%253D%253D&md5=398ba7a04c67671459a16bc4fe9a09b1Glutathione conjugation as a bioactivation reactionvan Bladeren, P. J.Chemico-Biological Interactions (2000), 129 (1-2), 61-76CODEN: CBINA8; ISSN:0009-2797. (Elsevier Science Ireland Ltd.)A review and discussion with 86 refs. In general, glutathione conjugation is regarded as a detoxication reaction. However, depending on the properties of the substrate, bioactivation is also possible. Four types of activation reaction have been recognized: direct-acting compds., conjugates that are activated through cysteine conjugate beta-lyase, conjugates that are activated through redox cycling and lastly conjugates that release the original reactive parent compd. The glutathione S-transferases have three connections with the formation of bioactivated conjugates: they catalyze their formation in a no. of cases, they are the earliest available target for covalent binding by these conjugates and lastly, the parent alkylating agents are regularly involved in the induction of the enzymes. Individual susceptibility for each of these agents is detd. by individual transferase subunit compn. and methods are becoming available to assess this susceptibility.
- 224Drobnica, L.; Kristián, P.; Augustín, J. The Chemistry of the NCS Group. Cyanates Their Thio Derivatives; Patai, P., Ed.; Wiley: New York, 1977; Part 2, pp 1003– 1221 DOI: 10.1002/9780470771532.ch6 .
- 225Shibata, T.; Kimura, Y.; Mukai, A.; Mori, H.; Ito, S.; Asaka, Y.; Oe, S.; Tanaka, H.; Takahashi, T.; Uchida, K. Transthiocarbamoylation of Proteins by Thiolated Isothiocyanates. J. Biol. Chem. 2011, 286, 42150, DOI: 10.1074/jbc.M111.308049[Crossref], [PubMed], [CAS], Google Scholar225https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFKjsr7E&md5=95fec76a0cce811c4dcfce40e8512ec8Transthiocarbamoylation of Proteins by Thiolated IsothiocyanatesShibata, Takahiro; Kimura, Yuuki; Mukai, Akihiro; Mori, Hitoshi; Ito, Sohei; Asaka, Yukio; Oe, Sho; Tanaka, Hiroshi; Takahashi, Takashi; Uchida, KojiJournal of Biological Chemistry (2011), 286 (49), 42150-42161, S42150/1-S42150/13CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Isothiocyanates, membrane-permeable electrophiles that form adducts with thiols, have been suggested to have important medical benefits. Here we shed light on isothiocyanate-thiol conjugates and studied their electrophilic potential transferring an isothiocyanate moiety to cellular proteins. When we examd. the effect of sulfhydryl mols. on cellular response induced by 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analog of sulforaphane isolated from broccoli, we obsd. significant induction of heme oxygenase-1 by 6-HITC even in the presence of N-acetyl-L-cysteine or glutathione (GSH). In addn., the authentic 6-HITC-β-mercaptoethanol (6-HITC-ME) conjugate markedly up-regulated the enzyme expression, suggesting the electrophilic potential of thiolated isothiocyanates. To gain a chem. insight into the cellular response induced by thiolated isothiocyanates, we studied the occurrence of transthiocarbamoylation of sulfhydryl mols. by 6-HITC-ME and obsd. that, upon incubation of 6-HITC-ME with GSH, a single product corresponding to the GSH conjugate of 6-HITC was generated. To test the functional ability of thiolated isothiocyanates to thiocarbamoylate proteins in living cells, we designed a novel probe, combining an isothiocyanate-reactive group and an alkyne functionality, and revealed that the transthiocarbamoylation of proteins occurred in the cells upon exposure to 6-HITC-ME. The target of thiocarbamoylation included heat shock protein 90 β (Hsp90β), a chaperone ATPase of the Hsp90 family implicated in protein maturation and targeting. To identify the sites of the Hsp90β modification, we utilized nano-LC/MALDI-TOF MS/MS and suggested that a thiol group on the peptide contg. Cys-521 reacted with 6-HITC, resulting in a covalent adduct in a 6-HITC-treated recombinant Hsp90β in vitro. The site-selective binding to Cys-521 was supported by in silico modeling. Further study on the thiocarbamoylation of Hsp90β suggested that the formation of 6-HITC-Hsp90β conjugate might cause activation of heat shock factor-1, rapidly signaling a potential heat shock response. These data suggest that thiolated isothiocyanates are an active metabolite that could contribute to cellular responses through transthiocarbamoylation of cellular proteins.
- 226Nakamura, T.; Kawai, Y.; Kitamoto, N.; Osawa, T.; Kato, Y. Covalent Modification of Lysine Residues by Allyl Isothiocyanate in Physiological Conditions: Plausible Transformation of Isothiocyanate from Thiol to Amine. Chem. Res. Toxicol. 2009, 22 (3), 536– 542, DOI: 10.1021/tx8003906[ACS Full Text], [CAS], Google Scholar226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhvFGms7Y%253D&md5=19f9ae27e52e9c9bfde9c1e27cabac62Covalent Modification of Lysine Residues by Allyl Isothiocyanate in Physiological Conditions: Plausible Transformation of Isothiocyanate from Thiol to AmineNakamura, Toshiyuki; Kawai, Yoshichika; Kitamoto, Noritoshi; Osawa, Toshihiko; Kato, YojiChemical Research in Toxicology (2009), 22 (3), 536-542CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)We investigated the reactivity of allyl isothiocyanate (AITC) with amino groups under physiol. conditions. First, the chem. reaction of AITC with bovine serum albumin (BSA) was investigated. When BSA was incubated with AITC in a phosphate buffer (pH 7.4), the loss of Lys residues was obsd. Second, the Lys residue Nα-benzoyl-glycyl-L-lysine (BGK) was reacted with AITC in the buffer, and a novel peak was detected using high performance liq. chromatog. (HPLC). The peak was purified and identified as AITC-modified BGK with a N.vepsiln.-thiocarbamoyl linkage. However, a thiol residue is known to be a predominant target of an isothiocyanate (ITC). Although AITC may react with a thiol moiety in vivo, a thiocarbamoyl linkage between ITC and thiol is unstable, and an AITC mol. may be regenerated. To prove the plausible transformation of ITC from thiol to amine, synthetic AITC-conjugated Nα-acetyl-L-cysteine (NAC) was incubated with BGK at 37° in physiol. buffer, and the generation of AITC-Lys was analyzed. The loss of the AITC-NAC adduct corresponded to the formation of the AITC-BGK adduct. Furthermore, using a novel monoclonal antibody (A4C7mAb) specific for AITC-Lys, we found that the AITC-Lys residue was generated from the reaction between AITC-NAC and BSA. Although AITC preferentially reacts with thiol rather than with Lys, AITC can be liberated from thiols and can then react with amino groups. The ITC-Lys adduct may be a useful marker for ITC target mols.
- 227Kumari, V.; Dyba, M. A.; Holland, R. J.; Liang, Y.-H.; Singh, S. V.; Ji, X. Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical. PLoS One 2016, 11 (9), e0163821, DOI: 10.1371/journal.pone.0163821[Crossref], [PubMed], [CAS], Google Scholar227https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitFGqtrY%253D&md5=3e02a10dd6bb210a738c0cb61d2bc48fIrreversible inhibition of glutathione S-transferase by phenethyl isothiocyanate (PEITC), a dietary cancer chemopreventive phytochemicalKumari, Vandana; Dyba, Marzena A.; Holland, Ryan J.; Liang, Yu-He; Singh, Shivendra V.; Ji, XinhuaPLoS One (2016), 11 (9), e0163821/1-e0163821/12CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Dietary isothiocyanates abundant as glucosinolate precursors in many edible cruciferous vegetables are effective for prevention of cancer in chem.-induced and transgenic rodent models. Some of these agents, including phenethyl isothiocyanate (PEITC), have already advanced to clin. investigations. The primary route of isothiocyanate metab. is its conjugation with glutathione (GSH), a reaction catalyzed by glutathione S-transferase (GST). The pi class GST of subunit type 1 (hGSTP1) is much more effective than the alpha class GST of subunit type 1 (hGSTA1) in catalyzing the conjugation. Here, we report the crystal structures of hGSTP1 and hGSTA1 each in complex with the GSH adduct of PEITC. We find that PEITC also covalently modifies the cysteine side chains of GST, which irreversibly inhibits enzymic activity.
- 228Wilson, A. J.; Kerns, J. K.; Callahan, J. F.; Moody, C. J. Keap Calm, and Carry on Covalently. J. Med. Chem. 2013, 56 (19), 7463– 7476, DOI: 10.1021/jm400224q[ACS Full Text], [CAS], Google Scholar228https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVKqtbvE&md5=ea907fb2ccd8c28142ab4fa2776d0195Keap Calm, and Carry on CovalentlyWilson, Anthony J.; Kerns, Jeffrey K.; Callahan, James F.; Moody, Christopher J.Journal of Medicinal Chemistry (2013), 56 (19), 7463-7476CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. The Nrf2-Keap1 system plays a major role in cellular defense against oxidative stress. Upon exposure to electrophiles, the cysteine-rich protein Keap1 is covalently modified, and it is this modification of Keap1 that allows the accumulation and subsequent nuclear translocation of Nrf2 where it induces the transcription of over 100 protective genes. This mechanism can be exploited in drug discovery approaches to diseases such as chronic kidney disease (CKD), chronic obstructive pulmonary disease (COPD), asthma, and neurodegenerative diseases like multiple sclerosis (MS) and Parkinson's, utilizing the modification of Keap1 by electrophiles, compds. that would not normally be considered useful in drug discovery programs. This Perspective discusses the development of potential therapies based on potent electrophiles, such as isothiocyanates and Michael acceptors, that, far from being assocd. with toxic events, can actually initiate a range of beneficial protective pathways.
- 229Lewis, S. M.; Li, Y.; Catalano, M. J.; Laciak, A. R.; Singh, H.; Seiner, D. R.; Reilly, T. J.; Tanner, J. J.; Gates, K. S. Inactivation of Protein Tyrosine Phosphatases by Dietary Isothiocyanates. Bioorg. Med. Chem. Lett. 2015, 25 (20), 4549– 4552, DOI: 10.1016/j.bmcl.2015.08.065[Crossref], [PubMed], [CAS], Google Scholar229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVCnu7vF&md5=aaeef7137d097799f44c0d5b1030e709Inactivation of protein tyrosine phosphatases by dietary isothiocyanatesLewis, Sarah M.; Li, Ya; Catalano, Michael J.; Laciak, Adrian R.; Singh, Harkewal; Seiner, Derrick R.; Reilly, Thomas J.; Tanner, John J.; Gates, Kent S.Bioorganic & Medicinal Chemistry Letters (2015), 25 (20), 4549-4552CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Isothiocyanates are bioactive dietary phytochems. that react readily with protein thiol groups. We find that isothiocyanates are time-dependent inactivators of cysteine-dependent protein tyrosine phosphatases (PTPs). Rate consts. for the inactivation of PTP1B and SHP-2 by allyl isothiocyanate (AITC) and sulforaphane (SF) range from 2 to 16 M-1 s-1. Results in the context of PTP1B are consistent with a mechanism involving covalent, yet reversible, modification of the enzyme's active site cysteine residue.
- 230Cross, J. V.; Foss, F. W.; Rady, J. M.; Macdonald, T. L.; Templeton, D. J. The Isothiocyanate Class of Bioactive Nutrients Covalently Inhibit the MEKK1 Protein Kinase. BMC Cancer 2007, 7 (1), 183, DOI: 10.1186/1471-2407-7-183[Crossref], [PubMed], [CAS], Google Scholar230https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2snmvFKjug%253D%253D&md5=e17c3a1681930fa43ed36a5e1d5f72d3The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinaseCross Janet V; Foss Frank W; Rady Joshua M; Macdonald Timothy L; Templeton Dennis JBMC cancer (2007), 7 (), 183 ISSN:.BACKGROUND: Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. METHODS: The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. RESULTS: ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. CONCLUSION: These results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins.
- 231Mi, L.; Xiao, Z.; Hood, B. L.; Dakshanamurthy, S.; Wang, X.; Govind, S.; Conrads, T. P.; Veenstra, T. D.; Chung, F.-L. Covalent Binding to Tubulin by Isothiocyanates: A Mechanism of Cell Growth Arrest and Apoptosis. J. Biol. Chem. 2008, 283 (32), 22136– 22146, DOI: 10.1074/jbc.M802330200[Crossref], [PubMed], [CAS], Google Scholar231https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXpt1artLc%253D&md5=de1c9ebf6ac3fbb4631e2cd26bbf955eCovalent Binding to Tubulin by Isothiocyanates: a mechanism of cell growth arrest and apoptosisMi, Lixin; Xiao, Zhen; Hood, Brian L.; Dakshanamurthy, Sivanesan; Wang, Xiantao; Govind, Sudha; Conrads, Thomas P.; Veenstra, Timothy D.; Chung, Fung-LungJournal of Biological Chemistry (2008), 283 (32), 22136-22146CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochem. mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and mol. mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examd. whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymn. in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.
- 232Ouertatani-Sakouhi, H.; El-Turk, F.; Fauvet, B.; Roger, T.; Le Roy, D.; Karpinar, D. P.; Leng, L.; Bucala, R.; Zweckstetter, M.; Calandra, T.; Lashuel, H. A. A New Class of Isothiocyanate-Based Irreversible Inhibitors of Macrophage Migration Inhibitory Factor. Biochemistry 2009, 48 (41), 9858– 9870, DOI: 10.1021/bi900957e[ACS Full Text], [CAS], Google Scholar232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFOksrfJ&md5=740dd080eca41baa8b21d8f71e02682aA New Class of Isothiocyanate-Based Irreversible Inhibitors of Macrophage Migration Inhibitory FactorOuertatani-Sakouhi, Hajer; El-Turk, Farah; Fauvet, Bruno; Roger, Thierry; Le Roy, Didier; Karpinar, Damla Pinar; Leng, Lin; Bucala, Richard; Zweckstetter, Markus; Calandra, Thierry; Lashuel, Hilal A.Biochemistry (2009), 48 (41), 9858-9870CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biol. activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogs occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochem., biophys., and structural studies to det. the effect of BITC and its analogs on the conformational state, quaternary structure, catalytic activity, receptor binding, and biol. activity of MIF. Light scattering, anal. ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodn. stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a redn. in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochem. and biol. activities of MIF and present new opportunities for modulating MIF biol. activities in vivo.
- 233Brown, K. K.; Hampton, M. B. Biological Targets of Isothiocyanates. Biochim. Biophys. Acta, Gen. Subj. 2011, 1810 (9), 888– 894, DOI: 10.1016/j.bbagen.2011.06.004[Crossref], [PubMed], [CAS], Google Scholar233https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptlantrs%253D&md5=1d34ab0921044fe7e7aa0eee3622e964Biological targets of isothiocyanatesBrown, Kristin K.; Hampton, Mark B.Biochimica et Biophysica Acta, General Subjects (2011), 1810 (9), 888-894CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)A review. Isothiocyanates are phytochems. with a broad array of effects in biol. systems. Bioactivity includes the stimulation of cellular antioxidant systems, induction of apoptosis and interference with cytokine prodn. and activity. Epidemiol. evidence and exptl. studies indicate that naturally occurring isothiocyanates and synthetic derivs. have anti-cancer and anti-inflammatory properties. This review focuses on the mol. targets of isothiocyanates, and how target modification translates into a biol. response. Isothiocyanates may mediate their effects via direct protein modification or indirectly by disruption of redox homeostasis and increased thiol oxidn. Some target proteins have been identified, but in-depth searches with new techniques are needed to reveal novel targets. Site-directed mutagenesis and isothiocyanate structure-activity relationships will assist in detg. the biol. significance of specific modifications. General significance: Target identification is important for rational drug design and exploiting the therapeutic potential of isothiocyanates. It also provides insight into the diverse pathways that these compds. regulate.
- 234Pearson, R. J.; Blake, D. G.; Mezna, M.; Fischer, P. M.; Westwood, N. J.; McInnes, C. The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1. Cell Chem. Biol. 2018, 25, 1107– 1116, DOI: 10.1016/j.chembiol.2018.06.001[Crossref], [PubMed], [CAS], Google Scholar234https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlart7%252FE&md5=29ef6760403e0925cc2c142bc85b0fa8The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1Pearson, Russell J.; Blake, David G.; Mezna, Mokdad; Fischer, Peter M.; Westwood, Nicholas J.; McInnes, CampbellCell Chemical Biology (2018), 25 (9), 1107-1116.e4CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)The polo kinase family are important oncol. targets that act in regulating entry into and progression through mitosis. Structure-guided discovery of a new class of inhibitors of Polo-like kinase 1 (PLK1) catalytic activity that interact with Cys67 of the ATP binding site is described. Compds. contg. the benzothiazole N-oxide scaffold not only bind covalently to this residue, but are reversible inhibitors through the formation of Meisenheimer complexes. This mechanism of kinase inhibition results in compds. that can target PLK1 with high selectivity, while avoiding issues with irreversible covalent binding and interaction with other thiol-contg. mols. in the cell. Due to renewed interest in covalent drugs and the plethora of potential drug targets, these represent prototypes for the design of kinase inhibitory compds. that achieve high specificity through covalent interaction and yet still bind reversibly to the ATP cleft, a strategy that could be applied to avoid issues with conventional covalent binders.
- 235Federici, L.; Lo Sterzo, C.; Pezzola, S.; Di Matteo, A. D.; Scaloni, F.; Federici, G.; Caccuri, A. M. Structural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-Transferases. Cancer Res. 2009, 69 (20), 8025– 8034, DOI: 10.1158/0008-5472.CAN-09-1314[Crossref], [PubMed], [CAS], Google Scholar235https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1ynurfN&md5=2ead5fbde7543b8a7983908ceedaf57eStructural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-TransferasesFederici, Luca; Lo Sterzo, Carlo; Pezzola, Silvia; Di Matteo, Adele; Scaloni, Flavio; Federici, Giorgio; Caccuri, Anna MariaCancer Research (2009), 69 (20), 8025-8034CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)Glutathione S-transferases (GST) constitute a superfamily of enzymes with diversified functions including detoxification from xenobiotics. In many human cancers, Pi class GST (GSTP1-1) is overexpressed and contributes to multidrug resistance by conjugating chemotherapeutics. In addn., GSTP1-1 displays antiapoptotic activity by interacting with c-Jun NH2-terminal kinase, a key regulator of apoptosis. Therefore, GSTP1-1 is considered a promising target for pharmaceutical treatment. Recently, a potent inhibitor of GSTs, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), was identified and tested on several tumor cell lines demonstrating high antiproliferative activity. To establish the structural basis of NBDHEX activity, we detd. the crystal structure of NBDHEX bound to either GSTP1-1 or GSTM2-2 (mu class). NBDHEX in both cases binds to the H-site but occupies different positions. Furthermore, the compd. is covalently attached to the GSH sulfur in the GSTM2-2 crystal, forming a σ-complex, although it is bound but not conjugated in the GSTP1-1 crystal. Several differences in the H-sites of the two isoenzymes det. the higher affinity of NBDHEX for GSTM2-2 with respect to GSTP1-1. One such difference is the presence of Ile104 in GSTP1-1 close to the bound NBDHEX, whereas the corresponding position is occupied by an alanine in GSTM2-2. Mutation of Ile104 into valine is a frequent GSTP1-1 polymorphism and we show here that the Ile104Val and Ile104Ala variants display a 4-fold higher affinity for the compd. Remarkably, the GSTP1-1/Ile104Ala structure in complex with NBDHEX shows a considerable shift of the compd. inside the H-site. These data might be useful for the development of new anticancer compds.
- 236Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal, M.; Stroud, R. M.; Gordon, E. M.; Wells, J. A. Site-Directed Ligand Discovery. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (17), 9367– 9372, DOI: 10.1073/pnas.97.17.9367[Crossref], [PubMed], [CAS], Google Scholar236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmtVensr8%253D&md5=fef57a0d21af9bc7f6f1f4bf8c023ae5Site-directed ligand discoveryErlanson, Daniel A.; Braisted, Andrew C.; Raphael, Darren R.; Randal, Mike; Stroud, Robert M.; Gordon, Eric M.; Wells, James A.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (17), 9367-9372CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We report a strategy (called "tethering") to discover low mol. wt. ligands (≈250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-contg. mols. (≈1,200 compds.) at concns. typically used in drug screening (10 to 200 μM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metab. with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (Ki≈1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallog. structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.
- 237Zong, L.; Bartolami, E.; Abegg, D.; Adibekian, A.; Sakai, N.; Matile, S. Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension. ACS Cent. Sci. 2017, 3 (5), 449– 453, DOI: 10.1021/acscentsci.7b00080[ACS Full Text], [CAS], Google Scholar237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsFOrsL4%253D&md5=6f68eab2bcb1b6100fb5970d1ee88a06Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest TensionZong, Lili; Bartolami, Eline; Abegg, Daniel; Adibekian, Alexander; Sakai, Naomi; Matile, StefanACS Central Science (2017), 3 (5), 449-453CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)The disulfide dihedral angle in epidithiodiketopiperazines (ETPs) is near 0°. Application of this highest possible ring tension to strain-promoted thiol-mediated uptake results in efficient delivery to the cytosol and nucleus. Compared to the previous best asparagusic acid (AspA), ring-opening disulfide exchange with ETPs occurs more efficiently even with nonactivated thiols, and the resulting thiols exchange rapidly with nonactivated disulfides. ETP-mediated cellular uptake is more than 20 times more efficient compared to AspA, occurs without endosomal capture, depends on temp., and is "unstoppable" by inhibitors of endocytosis and conventional thiol-mediated uptake, including siRNA against the transferrin receptor. These results suggest that ETP-mediated uptake not only maximizes delivery to the cytosol and nucleus but also opens the door to a new multitarget hopping mode of action.
- 238Tjin, C. C.; Otley, K. D.; Baguley, T. D.; Kurup, P.; Xu, J.; Nairn, A. C.; Lombroso, P. J.; Ellman, J. A. Glutathione-Responsive Selenosulfide Prodrugs as a Platform Strategy for Potent and Selective Mechanism-Based Inhibition of Protein Tyrosine Phosphatases. ACS Cent. Sci. 2017, 3 (12), 1322– 1328, DOI: 10.1021/acscentsci.7b00486[ACS Full Text], [CAS], Google Scholar238https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWnsrfN&md5=a1eff343feab9afb7b79628bf5efa034Glutathione-Responsive Selenosulfide Prodrugs as a Platform Strategy for Potent and Selective Mechanism-Based Inhibition of Protein Tyrosine PhosphatasesTjin, Caroline Chandra; Otley, Kate D.; Baguley, Tyler D.; Kurup, Pradeep; Xu, Jian; Nairn, Angus C.; Lombroso, Paul J.; Ellman, Jonathan A.ACS Central Science (2017), 3 (12), 1322-1328CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Dysregulation of protein tyrosine phosphorylation has been implicated in a no. of human diseases, including cancer, diabetes, and neurodegenerative disease. As a result of their essential role in regulating protein tyrosine phosphorylation levels, protein tyrosine phosphatases (PTPs) have emerged as important yet challenging therapeutic targets. Here the authors report on the development and application of a glutathione-responsive motif to facilitate the efficient intracellular delivery of a novel class of selenosulfide phosphatase inhibitors for the selective active site directed inhibition of the targeted PTP by selenosulfide exchange with the active site cysteine. The strategy leverages the large difference in extracellular and intracellular glutathione levels to deliver selenosulfide phosphatase inhibitors to cells. As an initial exploration of the prodrug platform and the corresponding selenosulfide covalent inhibitor class, potent and selective inhibitors were developed for two therapeutically relevant PTP targets: the Mycobacterium tuberculosis virulence factor mPTPA and the CNS-specific tyrosine phosphatase, STtriatal-Enriched protein tyrosine Phosphatase (STEP). The lead selenosulfide inhibitors enable potent and selective inhibition of their resp. targets over a panel of human PTPs and a representative cysteine protease. Kinetic parameters of the inhibitors were characterized, including reversibility of inhibition and rapid rate of GSH exchange at intracellular GSH concns. Addnl., active site covalent inhibitor-labeling with an mPTPA inhibitor was rigorously confirmed by mass spectrometry and cellular activity was demonstrated with a STEP prodrug inhibitor in cortical neurons.
- 239Weichert, D.; Gmeiner, P. Covalent Molecular Probes for Class A G Protein-Coupled Receptors: Advances and Applications. ACS Chem. Biol. 2015, 10 (6), 1376– 1386, DOI: 10.1021/acschembio.5b00070[ACS Full Text], [CAS], Google Scholar239https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtlamsrw%253D&md5=1defbb0685a38101822c936eac6ba2f1Covalent Molecular Probes for Class A G Protein-Coupled Receptors: Advances and ApplicationsWeichert, Dietmar; Gmeiner, PeterACS Chemical Biology (2015), 10 (6), 1376-1386CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A review. Covalent modification of G protein-coupled receptors (GPCRs) by employing specific mol. probes has for decades provided a successful strategy to facilitate the elucidation of the structure and function of this pharmacol. important class of membrane proteins. The ligands typically comprise a pharmacophore that generates affinity for a given GPCR and contain a reactive functionality that may form a covalent bond with a suitably positioned amino acid residue. Covalent ligands have been successfully applied to circumvent poor affinity of compds. when stable labeling of receptor populations was required, and they have been used in the isolation, purifn., and pharmacol. characterization of specific subtypes of GPCRs. Recently, structural studies have demonstrated that covalent mol. probes are effective at stabilizing GPCRs to obtain X-ray crystal structures, thus providing valuable insights for the development of novel therapeutics. Herein, we review covalently binding mol. probes for class A GPCRs with a focus on ligands comprising crosslinking groups that do not require photoactivation and further highlight their significant and diverse applications.
- 240Rosenbaum, D. M.; Zhang, C.; Lyons, J. A.; Holl, R.; Aragao, D.; Arlow, D. H.; Rasmussen, S. G. F.; Choi, H.-J.; DeVree, B. T.; Sunahara, R. K.; Chae, P. S.; Gellman, S. H.; Dror, R. O.; Shaw, D. E.; Weis, W. I.; Caffrey, M.; Gmeiner, P.; Kobilka, B. K. Structure and Function of an Irreversible Agonist-Β2 Adrenoceptor Complex. Nature 2011, 469 (7329), 236– 240, DOI: 10.1038/nature09665[Crossref], [PubMed], [CAS], Google Scholar240https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkvFehsQ%253D%253D&md5=8629b93e43fb692395e2aa6f8bb011a9Structure and function of an irreversible agonist-β2 adrenoceptor complexRosenbaum, Daniel M.; Zhang, Cheng; Lyons, Joseph A.; Holl, Ralph; Aragao, David; Arlow, Daniel H.; Rasmussen, Soren G. F.; Choi, Hee-Jung; DeVree, Brian T.; Sunahara, Roger K.; Chae, Pil Seok; Gellman, Samuel H.; Dror, Ron O.; Shaw, David E.; Weis, William I.; Caffrey, Martin; Gmeiner, Peter; Kobilka, Brian K.Nature (London, United Kingdom) (2011), 469 (7329), 236-240CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biol. function by initiating cellular signaling in response to chem. diverse agonists. Despite recent progress in the structural biol. of GPCRs, the mol. basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystn. of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β2 adrenergic receptor (β2AR) as a guide, we designed a β2AR agonist that can be covalently tethered to a specific site on the receptor through a disulfide bond. The covalent β2AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystd. a covalent agonist-bound β2AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and detd. its structure at 3.5 Å resoln. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) mol. dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.
- 241Schwalbe, T.; Kaindl, J.; Hübner, H.; Gmeiner, P. Potent Haloperidol Derivatives Covalently Binding to the Dopamine D2 Receptor. Bioorg. Med. Chem. 2017, 25 (19), 5084– 5094, DOI: 10.1016/j.bmc.2017.06.034[Crossref], [PubMed], [CAS], Google Scholar241https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVyntb7O&md5=c7357c0703b5ee0ee9c7ba496fcef4e4Potent haloperidol derivatives covalently binding to the dopamine D2 receptorSchwalbe, Tobias; Kaindl, Jonas; Huebner, Harald; Gmeiner, PeterBioorganic & Medicinal Chemistry (2017), 25 (19), 5084-5094CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)The dopamine D2 receptor (D2R) is a common drug target for the treatment of a variety of neurol. disorders including schizophrenia. Structure based design of subtype selective D2R antagonists requires high resoln. crystal structures of the receptor and pharmacol. tools promoting a better understanding of the protein-ligand interactions. Recently, we reported the development of a chem. activated dopamine deriv. (FAUC150) designed to covalently bind the L94C mutant of the dopamine D2 receptor. Using FAUC150 as a template, we elaborated the design and synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming covalent D2R-ligand complexes. The disulfide- and Michael acceptor-functionalized compds. showed significant receptor affinity and an irreversible binding profile in radioligand depletion expts.
- 242Liu, Y.; Xie, Z.; Zhao, D.; Zhu, J.; Mao, F.; Tang, S.; Xu, H.; Luo, C.; Geng, M.; Huang, M.; Li, J. Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) Inhibitors. J. Med. Chem. 2017, 60 (6), 2227– 2244, DOI: 10.1021/acs.jmedchem.6b01245[ACS Full Text], [CAS], Google Scholar242https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjtlyjtr0%253D&md5=7529ce2fc9d1a138a8d1405a23f7e4e8Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) InhibitorsLiu, Yifu; Xie, Zuoquan; Zhao, Dan; Zhu, Jin; Mao, Fei; Tang, Shuai; Xu, Hui; Luo, Cheng; Geng, Meiyu; Huang, Min; Li, JianJournal of Medicinal Chemistry (2017), 60 (6), 2227-2244CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Pyruvate dehydrogenase kinases (PDKs) are overexpressed in most cancer cells and are responsible for aberrant glucose metab. The authors previously described Bis(4-morpholinyl thiocarbonyl)-disulfide (JX06, 16) as the first covalent inhibitor of PDK1. Here, based on the scaffold of JX06, the authors identify two novel types of disulfide-based PDK1 inhibitors. The most potent analog, bis(1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3a), effectively inhibits PDK1 both at the mol. (kinact/Ki = 4.17 × 103 M-1s-1) and the cellular level (down to 0.1 μM). In contrast to JX06, 3a is a potent and subtype-selective inhibitor of PDK1 with >40-fold selectivity for PDK2-4. Compd. 3a also significantly alters glucose metabolic pathways in A549 cells by decreasing ECAR and increasing ROS. Moreover, in the xenograft models, 3a shows significant antitumor activity with no neg. effect to the mice wt. Collectively, these data demonstrate that 3a may be an excellent lead compd. for the treatment of cancer as a first-generation subtype-selective and covalent PDK1 inhibitor.
- 243Napolitano, L.; Scalise, M.; Koyioni, M.; Koutentis, P.; Catto, M.; Eberini, I.; Parravicini, C.; Palazzolo, L.; Pisani, L.; Galluccio, M.; Console, L.; Carotti, A.; Indiveri, C. Potent Inhibitors of Human LAT1 (SLC7A5) Transporter Based on Dithiazole and Dithiazine Compounds for Development of Anticancer Drugs. Biochem. Pharmacol. 2017, 143, 39– 52, DOI: 10.1016/j.bcp.2017.07.006[Crossref], [PubMed], [CAS], Google Scholar243https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFOlsL3I&md5=5953170d4a704a9c546775f784ef9fc5Potent inhibitors of human LAT1 (SLC7A5) transporter based on dithiazole and dithiazine compounds for development of anticancer drugsNapolitano, Lara; Scalise, Mariafrancesca; Koyioni, Maria; Koutentis, Panayiotis; Catto, Marco; Eberini, Ivano; Parravicini, Chiara; Palazzolo, Luca; Pisani, Leonardo; Galluccio, Michele; Console, Lara; Carotti, Angelo; Indiveri, CesareBiochemical Pharmacology (Amsterdam, Netherlands) (2017), 143 (), 39-52CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)The LAT1 transporter is acknowledged as a pharmacol. target of tumors since it is strongly overexpressed in many human cancers. The purpose of this work was to find novel compds. exhibiting potent and prolonged inhibition of the transporter. To this aim, compds. based on dithiazole and dithiazine scaffold have been screened in the proteoliposome exptl. model. Inhibition was tested on the antiport catalyzed by hLAT1 as transport of extraliposomal [3H]histidine in exchange with intraliposomal histidine. Out of 59 compds. tested, 8 compds., showing an inhibition higher than 90% at 100 μM concn., were subjected to dose-response anal. Two of them exhibited IC50 lower than 1 μM. Inhibition kinetics, performed on the two best inhibitors, indicated a mixed type of inhibition with respect to the substrate. Furthermore, inhibition of the transporter was still present after removal of the compds. from the reaction mixt., but was reversed on addn. of dithioerythritol, a S-S reducing agent, indicating the formation of disulfide(s) between the compds. and the protein. Mol. docking of the two best inhibitors on the hLAT1 homol. structural model, highlighted interaction with the substrate binding site and formation of a covalent bond with the residue C407. Indeed, the inhibition was impaired in the hLAT1 mutant C407A confirming the involvement of that Cys residue. Treatment of SiHa cells expressing hLAT1 at relatively high level, with the two most potent inhibitors led to cell death which was not obsd. after treatment with a compd. exhibiting very poor inhibitory effect.
- 244Nagy, P. Kinetics and Mechanisms of Thiol–Disulfide Exchange Covering Direct Substitution and Thiol Oxidation-Mediated Pathways. Antioxid. Redox Signaling 2013, 18 (13), 1623– 1641, DOI: 10.1089/ars.2012.4973[Crossref], [PubMed], [CAS], Google Scholar244https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXkvFOjtLo%253D&md5=3e1793928a0494099aabab96413bcf8bKinetics and Mechanisms of Thiol-Disulfide Exchange Covering Direct Substitution and Thiol Oxidation-Mediated PathwaysNagy, PeterAntioxidants & Redox Signaling (2013), 18 (13), 1623-1641CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)A review. Significance: Disulfides are important building blocks in the secondary and tertiary structures of proteins, serving as inter- and intra-subunit cross links. Disulfides are also the major products of thiol oxidn., a process that has primary roles in defense mechanisms against oxidative stress and in redox regulation of cell signaling. Although disulfides are relatively stable, their redn., isomerisation, and interconversion as well as their prodn. reactions are catalyzed by delicate enzyme machineries, providing a dynamic system in biol. Redox homeostasis, a thermodn. parameter that dets. which reactions can occur in cellular compartments, is also balanced by the thiol-disulfide pool. However, it is the kinetic properties of the reactions that best represent cell dynamics, because the partitioning of the possible reactions depends on kinetic parameters. Crit. Issues: This review is focused on the kinetics and mechanisms of thiol-disulfide substitution and redox reactions. It summarizes the challenges and advances that are assocd. with kinetic investigations in small mol. and enzymic systems from a rigorous chem. perspective using biol. examples. The most important parameters that influence reaction rates are discussed in detail. Recent Advances and Future Directions: Kinetic studies of proteins are more challenging than small mols., and quite often investigators are forced to sacrifice the rigor of the exptl. approach to obtain the important kinetic and mechanistic information. However, recent technol. advances allow a more comprehensive anal. of enzymic systems via using the systematic kinetics app. that was developed for small mol. reactions, which is expected to provide further insight into the cell's machinery. Antioxid. Redox Signal. 18, 1623-1641.
- 245Go, Y.-M.; Jones, D. P. Thiol/Disulfide Redox States in Signaling and Sensing. Crit. Rev. Biochem. Mol. Biol. 2013, 48 (2), 173– 181, DOI: 10.3109/10409238.2013.764840[Crossref], [PubMed], [CAS], Google Scholar245https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXkslWntbc%253D&md5=3e9e065adbd2a5b7af0bdd904c5f1719Thiol/disulfide redox states in signaling and sensingGo, Young-Mi; Jones, Dean P.Critical Reviews in Biochemistry and Molecular Biology (2013), 48 (2), 173-181CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare)A review. Rapid advances in redox systems biol. are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol-disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady states displaced from thermodn. equil., that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biol. systems and the knowledge base available to support development of integrated redox systems biol. models to better understand the function and dysfunction of thiol-disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiol. function. Advances in redox proteomics show that, in addn. to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biol. functions. These advances provide a framework for translation of redox systems biol. concepts to practical use in understanding and treating human disease. Biol. responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities.
- 246García-Santamarina, S.; Boronat, S.; Hidalgo, E. Reversible Cysteine Oxidation in Hydrogen Peroxide Sensing and Signal Transduction. Biochemistry 2014, 53 (16), 2560– 2580, DOI: 10.1021/bi401700f[ACS Full Text], [CAS], Google Scholar246https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmt1Gju78%253D&md5=3e5ea91a2214801e4b84066acf71505cReversible Cysteine Oxidation in Hydrogen Peroxide Sensing and Signal TransductionGarcia-Santamarina, Sarela; Boronat, Susanna; Hidalgo, ElenaBiochemistry (2014), 53 (16), 2560-2580CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. Activation of redox cascades through hydrogen peroxide-mediated reversible cysteine oxidn. is a major mechanism for intracellular signaling. Understanding why some cysteine residues are specifically oxidized, in competition with other proximal cysteine residues and in the presence of strong redox buffers, is therefore crucial for understanding redox signaling. In this review, the authors explore the recent advances in thiol-redox chem. linked to signaling. The authors describe the last findings in the field of redox sensors, those that are naturally present in different model organisms as well as those that have been engineered to quantify intracellular hydrogen peroxide concns. Finally, the authors provide a summary of the newest approaches developed to study reversible cysteine oxidn. at the proteomic level.
- 247Gupta, V.; Carroll, K. S. Profiling the Reactivity of Cyclic C-Nucleophiles towards Electrophilic Sulfur in Cysteine Sulfenic Acid. Chem. Sci. 2016, 7 (1), 400– 415, DOI: 10.1039/C5SC02569A[Crossref], [PubMed], [CAS], Google Scholar247https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1WltrnN&md5=249ce8ace72189fc42d75515333afe08Profiling the reactivity of cyclic C-nucleophiles towards electrophilic sulfur in cysteine sulfenic acidGupta, Vinayak; Carroll, Kate S.Chemical Science (2016), 7 (1), 400-415CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Oxidn. of a protein cysteine thiol to sulfenic acid, termed S-sulfenylation, is a reversible post-translational modification that plays a crucial role in regulating protein function and is correlated with disease states. The majority of reaction-based small mol. and immunochem. probes used for detecting sulfenic acids are based on the 5,5-dimethyl-1,3-cyclohexanedione (dimedone) scaffold, which is selective, but suffers from low reactivity. In addn., mechanistic details and features that diminish or enhance nucleophile reactivity remain largely unknown. A significant hurdle to resolving the aforementioned issues has been the chem. unstable nature of small-mol. sulfenic acid models. Herein, we report a facile mass spectrometry-based assay and repurposed dipeptide-based model to screen a library of cyclic C-nucleophiles for reactivity with sulfenic acid under aq. conditions. Obsd. rate consts. for ∼100 cyclic C-nucleophiles were obtained and, from this collection, we have identified novel compds. with more than 200-fold enhanced reactivity, as compared to dimedone. The increase in reactivity and retention of selectivity of these C-nucleophiles were validated in secondary assays, including a protein model for sulfenic acid. Together, this work represents a significant step toward developing new chem. reporters for detecting protein S-sulfenylation with superior kinetic resoln. The enhanced rates and varied compn. of the C-nucleophiles should enable more comprehensive analyses of the sulfenome and serve as the foundation for reversible or irreversible nucleophilic covalent inhibitors that target oxidized cysteine residues in therapeutically important proteins.
- 248Truong, T. H.; Carroll, K. S. Redox Regulation of Epidermal Growth Factor Receptor Signaling through Cysteine Oxidation. Biochemistry 2012, 51 (50), 9954– 9965, DOI: 10.1021/bi301441e[ACS Full Text], [CAS], Google Scholar248https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhslelsr7O&md5=80ce6e8d25c413b3e4040fd391f87555Redox regulation of epidermal growth factor receptor signaling through cysteine oxidationTruong, Thu H.; Carroll, Kate S.Biochemistry (2012), 51 (50), 9954-9965CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. Epidermal growth factor receptor (EGFR) exemplifies the family of receptor tyrosine kinases that mediate numerous cellular processes, including growth, proliferation, and differentiation. Moreover, gene amplification and EGFR mutations have been identified in a no. of human malignancies, making this receptor an important target for the development of anticancer drugs. In addn. to ligand-dependent activation and concomitant tyrosine phosphorylation, EGFR stimulation results in the localized generation of H2O2 by NADPH-dependent oxidases. In turn, H2O2 functions as a secondary messenger to regulate intracellular signaling cascades, largely through the modification of specific cysteine residues within redox-sensitive protein targets, including Cys797 in the EGFR active site. In this review, we highlight recent advances in our understanding of the mechanisms that underlie redox regulation of EGFR signaling and how these discoveries may form the basis for the development of new therapeutic strategies for targeting this and other H2O2-modulated pathways.
- 249Garcia, F. J.; Carroll, K. S. Redox-Based Probes as Tools to Monitor Oxidized Protein Tyrosine Phosphatases in Living Cells. Eur. J. Med. Chem. 2014, 88, 28– 33, DOI: 10.1016/j.ejmech.2014.06.040[Crossref], [PubMed], [CAS], Google Scholar249https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVOju7vP&md5=221c3c815cb7fd727ce86ea5b88a0c06Redox-based probes as tools to monitor oxidized protein tyrosine phosphatases in living cellsGarcia, Francisco J.; Carroll, Kate S.European Journal of Medicinal Chemistry (2014), 88 (), 28-33CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Reversible oxidn. of protein tyrosine phosphatases (PTPs) has emerged as an important regulatory mechanism whereby reactive oxygen species (ROS) inactivates the PTP and promotes phosphorylation and induction of the signaling cascade. The lack of sensitive and robust methods to directly detect oxidized PTPs has made it difficult to understand the effects that PTP oxidative inactivation play in redox signaling. We report the use of redox-based probes to directly detect oxidized PTPs in a cellular context, which highlights the importance of direct approaches to assist in the study of physiol. and pathophysiol. PTP activity in redox regulation. We also demonstrate, as a proof-of-concept, that these redox-based probes serve as prototypes for the design and development of a new class of inhibitors for phosphatases. We envision a nucleophile reacting with the oxidized inactive catalytic cysteine to generate an irreversible thioether adduct which prevents the phosphatase from being reactivated and ultimately fortifies the signaling cascade. Our results reveal the potential of translation of our redox-based probes, which are used to understand redox cell circuitry and disease biol., to small-mol. nucleophile-based inhibitors, which may treat diseases assocd. with redox stress. This may have implications in the treatment of type 2 diabetes and cancer.
- 250Alcock, L. J.; Farrell, K. D.; Akol, M. T.; Jones, G. H.; Tierney, M. M.; Kramer, H. B.; Pukala, T. L.; Bernardes, G. J. L.; Perkins, M. V.; Chalker, J. M. Norbornene Probes for the Study of Cysteine Oxidation. Tetrahedron 2018, 74 (12), 1220– 1228, DOI: 10.1016/j.tet.2017.11.011[Crossref], [CAS], Google Scholar250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVWqurbE&md5=bcf72e863b0040d346a907351c72409fNorbornene probes for the study of cysteine oxidationAlcock, Lisa J.; Farrell, Kyle D.; Akol, Mawey T.; Jones, Gregory H.; Tierney, Matthew M.; Kramer, Holger B.; Pukala, Tara L.; Bernardes, Goncalo J. L.; Perkins, Michael V.; Chalker, Justin M.Tetrahedron (2018), 74 (12), 1220-1228CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)Cysteine residues on proteins can react with cellular oxidants such as hydrogen peroxide. While this process is important for scavenging excess reactive oxygen species, the products of this oxidn. may also mediate cell signaling. To understand the role of cysteine oxidn. in biol., selective probes are required to detect and quantify its occurrence. Cysteine oxidn. products such as sulfenic acids are sometimes unstable and therefore short-lived. If such cysteine derivs. are to be analyzed, rapid reaction with the probe is required. Here the authors introduce norbornene derivs. as probes for cysteine oxidn., and demonstrate their ability to trap sulfenic acids. The synthesis of norbornene derivs. contg. alkyne or biotin affinity tags are also reported to facilitate the use of these probes in chem. biol. and proteomics.
- 251Poole, T. H.; Reisz, J. A.; Zhao, W.; Poole, L. B.; Furdui, C. M.; King, S. B. Strained Cycloalkynes as New Protein Sulfenic Acid Traps. J. Am. Chem. Soc. 2014, 136 (17), 6167– 6170, DOI: 10.1021/ja500364r[ACS Full Text], [CAS], Google Scholar251https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtVegtL0%253D&md5=af7b13df6e6bd4101306df5be38bd88aStrained Cycloalkynes as New Protein Sulfenic Acid TrapsPoole, Thomas H.; Reisz, Julie A.; Zhao, Weiling; Poole, Leslie B.; Furdui, Cristina M.; King, S. BruceJournal of the American Chemical Society (2014), 136 (17), 6167-6170CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein sulfenic acids are formed by the reaction of biol. relevant reactive oxygen species with protein thiols. Sulfenic acid formation modulates the function of enzymes and transcription factors either directly or through the subsequent formation of protein disulfide bonds. Identifying the site, timing, and conditions of protein sulfenic acid formation remains crucial to understanding cellular redox regulation. Current methods for trapping and analyzing sulfenic acids involve the use of dimedone and other nucleophilic 1,3-dicarbonyl probes that form covalent adducts with cysteine-derived protein sulfenic acids. As a mechanistic alternative, the present study describes highly strained bicyclo[6.1.0]nonyne (BCN) derivs. as concerted traps of sulfenic acids. These strained cycloalkynes react efficiently with sulfenic acids in proteins and small mols. yielding stable alkenyl sulfoxide products at rates >100x greater than 1,3-dicarbonyl reagents enabling kinetic competition with physiol. sulfur chem. Similar to the 1,3-dicarbonyl reagents, the BCN compds. distinguish the sulfenic acid oxoform from the thiol, disulfide, sulfinic acid, and S-nitrosated forms of cysteine while displaying an acceptable cell toxicity profile. The enhanced rates demonstrated by these strained alkynes identify them as new bioorthogonal probes that should facilitate the discovery of previously unknown sulfenic acid sites and their parent proteins.
- 252Gupta, V.; Carroll, K. S. Rational Design of Reversible and Irreversible Cysteine Sulfenic Acid-Targeted Linear C-Nucleophiles. Chem. Commun. 2016, 52 (16), 3414– 3417, DOI: 10.1039/C6CC00228E[Crossref], [PubMed], [CAS], Google Scholar252https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtl2ns7g%253D&md5=4279f7d9100cadfe4747bfebf97c2845Rational design of reversible and irreversible cysteine sulfenic acid-targeted linear C-nucleophilesGupta, Vinayak; Carroll, Kate S.Chemical Communications (Cambridge, United Kingdom) (2016), 52 (16), 3414-3417CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Concerns about off-target effects has motivated the development of reversible covalent inhibition strategies for targeting cysteine. However, such strategies have not been reported for the unique cysteine oxoform, sulfenic acid. Herein, we have designed and identified linear C-nucleophiles that react selectively with cysteine sulfenic acid. The resulting thioether adducts exhibit reversibility ranging from minutes to days under reducing conditions, showing the feasibility of tuning C-nucleophile reactivity across a wide range of time scales.
- 253Forman, H. J.; Davies, M. J.; Krämer, A. C.; Miotto, G.; Zaccarin, M.; Zhang, H.; Ursini, F. Protein Cysteine Oxidation in Redox Signaling: Caveats on Sulfenic Acid Detection and Quantification. Arch. Biochem. Biophys. 2017, 617, 26– 37, DOI: 10.1016/j.abb.2016.09.013[Crossref], [PubMed], [CAS], Google Scholar253https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1WntrjF&md5=d37c64fc4beecbb6d8349255064f6236Protein cysteine oxidation in redox signaling: Caveats on sulfenic acid detection and quantificationForman, Henry Jay; Davies, Michael J.; Kramer, Anna C.; Miotto, Giovanni; Zaccarin, Mattia; Zhang, Hongqiao; Ursini, FulvioArchives of Biochemistry and Biophysics (2017), 617 (), 26-37CODEN: ABBIA4; ISSN:0003-9861. (Elsevier B.V.)Oxidn. of crit. signaling protein cysteines regulated by H2O2 has been considered to involve sulfenic acid (RSOH) formation. RSOH may subsequently form either a sulfenyl amide (RSNHR') with a neighboring amide, or a mixed disulfide (RSSR') with another protein cysteine or glutathione. Previous studies have claimed that RSOH can be detected as an adduct (e.g., with 5,5-dimethylcyclohexane-1,3-dione; dimedone). Here, kinetic data are discussed which indicate that few proteins can form RSOH under physiol. signaling conditions. We also present exptl. evidence that indicates that (1) dimedone reacts rapidly with sulfenyl amides, and more rapidly than with sulfenic acids, and (2) that disulfides can react reversibly with amides to form sulfenyl amides. As some proteins are more stable as the sulfenyl amide than as a glutathionylated species, the former may account for some of the species previously identified as the "sulfenome" - the cellular complement of reversibly-oxidized thiol proteins generated via sulfenic acids.
- 254Gupta, V.; Yang, J.; Liebler, D. C.; Carroll, K. S. Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles. J. Am. Chem. Soc. 2017, 139 (15), 5588– 5595, DOI: 10.1021/jacs.7b01791[ACS Full Text], [CAS], Google Scholar254https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXltlCmsbY%253D&md5=ac0b22e7270f452df5851033ae73e126Diverse Redoxome Reactivity Profiles of Carbon NucleophilesGupta, Vinayak; Yang, Jing; Liebler, Daniel C.; Carroll, Kate S.Journal of the American Chemical Society (2017), 139 (15), 5588-5595CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this process is the identification of signaling pathways (or receptors) specific to (or overexpressed in) disease cells. In this context, fragment-based ligand discovery (FBLD) has significantly expanded our view of the ligandable proteome and affords tool compds. for biol. inquiry. To date, such covalent ligand discovery has almost exclusively employed cysteine-reactive small-mol. fragments. However, functional cysteine residues in proteins are often redox-sensitive and can undergo oxidn. in cells. Such reactions are particularly relevant in diseases, like cancer, which are linked to excessive prodn. of reactive oxygen species. Once oxidized, the sulfur atom of cysteine is much less reactive toward electrophilic groups used in the traditional FBLD paradigm. To address this limitation, we recently developed a novel library of diverse carbon-based nucleophile fragments that react selectively with cysteine sulfenic acid formed in proteins via oxidn. or hydrolysis reactions. Here, we report anal. of sulfenic acid-reactive C-nucleophile fragments screened against a colon cancer cell proteome. Covalent ligands were identified for >1280 S-sulfenylated cysteines present in "druggable" proteins and orphan targets, revealing disparate reactivity profiles and target preferences. Among the unique ligand-protein interactions identified was that of a pyrrolidinedione nucleophile that reacted preferentially with protein tyrosine phosphatases. Fragment-based covalent ligand discovery with C-nucleophiles affords an expansive snapshot of the ligandable "redoxome" with significant implications for covalent inhibitor pharmacol. and also affords new chem. tools to investigate redox-regulation of protein function.
- 255Holliday, G. L.; Mitchell, J. B. O.; Thornton, J. M. Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis. J. Mol. Biol. 2009, 390 (3), 560– 577, DOI: 10.1016/j.jmb.2009.05.015[Crossref], [PubMed], [CAS], Google Scholar255https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnsl2itbc%253D&md5=15be47b78d45b7d3c8f393e3bd20fc20Understanding the functional roles of amino acid residues in enzyme catalysisHolliday, Gemma L.; Mitchell, John B. O.; Thornton, Janet M.Journal of Molecular Biology (2009), 390 (3), 560-577CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The MACiE database contains 223 distinct step-wise enzyme reaction mechanisms and holds representatives from each EC sub-subclass where there is a crystal structure and sufficient evidence in the literature to support a mechanism. Each catalytic step of every reaction sequence in MACiE is fully annotated so that it includes the function of the catalytic residues involved in the reaction and the mechanism by which substrates are transformed into products. Using MACiE as a knowledge base, the authors have seen that the top 10 most catalytic residues are His, Asp, Glu, Lys, Cys, Arg, Ser, Thr, Tyr, and Trp. Of these, only 7 (Cys, His, Asp, Lys, Ser, Thr, and Tyr) dominate catalysis and provide essentially 5 functional roles that are essential. Stabilization is the most common and essential role for all classes of enzyme, followed by general acid/base (proton acceptor and proton donor) functionality, with nucleophilic addn. following closely behind (nucleophile and nucleofuge). The authors investigated the occurrence of these residues in MACiE and the Catalytic Site Atlas and found that, as expected, certain residue types were assocd. with each functional role, with some residue types able to perform diverse roles. In addn., it was seen that different EC classes of enzyme had a tendency to employ different residues for catalysis. Further, the authors showed that while the differences between EC classes in catalytic residue compn. were not immediately obvious from the general classes of Ingold mechanisms, there was some weak correlation between the mechanisms involved in a given EC class and the functions that the catalytic amino acid residues were performing. The anal. presented here provides a valuable insight into the functional roles of catalytic amino acid residues, which may have applications in many aspects of enzymol., from the design of novel enzymes to the prediction and validation of enzyme reaction mechanisms.
- 256Platzer, G.; Okon, M.; McIntosh, L. P. PH-Dependent Random Coil 1H, 13C, and 15N Chemical Shifts of the Ionizable Amino Acids: A Guide for Protein PKa Measurements. J. Biomol. NMR 2014, 60 (2–3), 109– 129, DOI: 10.1007/s10858-014-9862-y[Crossref], [PubMed], [CAS], Google Scholar256https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFOqtbjM&md5=196361f2a4e7a2ba970a50844c578cdbpH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pKa measurementsPlatzer, Gerald; Okon, Mark; McIntosh, Lawrence P.Journal of Biomolecular NMR (2014), 60 (2-3), 109-129CODEN: JBNME9; ISSN:0925-2738. (Springer)The pKa values and charge states of ionizable residues in polypeptides and proteins are frequently detd. via NMR-monitored pH titrns. To aid the interpretation of the resulting titrn. data, we have measured the pH-dependent chem. shifts of nearly all the 1H, 13C, and 15N nuclei in the seven common ionizable amino acids (X = Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, resp. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor ref. chem. shifts to help guide protein pKa measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chem. shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain 15N nuclei of His, Lys, and Arg in their pos.-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary exptl. evidence for defining the ionization states of protein residues.
- 257Isom, D. G.; Castañeda, C. A.; Cannon, B. R.; Garcia-Moreno, B. E. Large Shifts in PKa Values of Lysine Residues Buried inside a Protein. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (13), 5260– 5265, DOI: 10.1073/pnas.1010750108[Crossref], [PubMed], [CAS], Google Scholar257https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkslOmt7c%253D&md5=aefb9e5d2cb9362ff63688fab588f7b0Large shifts in pKa values of lysine residues buried inside a proteinIsom, Daniel G.; Castaneda, Carlos A.; Cannon, Brian R.; Garcia-Moreno, Bertrand E.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (13), 5260-5265, S5260/1-S5260/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine mol. determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pKa values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pKa values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biol. H+ transport. The pKa values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pKa values varied significantly from location to location inside the protein. These data will enable an unprecedented examn. of the positional dependence of the dielec. response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function.
- 258Hacker, S. M.; Backus, K. M.; Lazear, M. R.; Forli, S.; Correia, B. E.; Cravatt, B. F. Global Profiling of Lysine Reactivity and Ligandability in the Human Proteome. Nat. Chem. 2017, 9 (12), 1181– 1190, DOI: 10.1038/nchem.2826[Crossref], [PubMed], [CAS], Google Scholar258https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1GrsrrN&md5=1dac8f079c1caffc8ae584291480fc61Global profiling of lysine reactivity and ligandability in the human proteomeHacker, Stephan M.; Backus, Keriann M.; Lazear, Michael R.; Forli, Stefano; Correia, Bruno E.; Cravatt, Benjamin F.Nature Chemistry (2017), 9 (12), 1181-1190CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, the authors report a chem. proteomic platform for the global and quant. anal. of lysine residues in native biol. systems. The authors have quantified, in total, >9000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small mols. The authors also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.
- 259Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Structural Determinants of Phosphoinositide 3-Kinase Inhibition by Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine. Mol. Cell 2000, 6 (4), 909– 919, DOI: 10.1016/S1097-2765(05)00089-4[Crossref], [PubMed], [CAS], Google Scholar259https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnvVegurY%253D&md5=5dd92f9b48c437e43c0023fdff10c1acStructural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporineWalker, Edward H.; Pacold, Michael E.; Perisic, Olga; Stephens, Len; Hawkins, Philip T.; Wymann, Matthias P.; Williams, Roger L.Molecular Cell (2000), 6 (4), 909-919CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)The specific phosphoinositide 3-kinase (P13K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallog. structures of P13K bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compds. fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compd. on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind P13K in remarkably different orientations that are related to each other by 1800 rotations. Staurosporine/P13K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific P13K inhibitors with therapeutic potential.
- 260Wymann, M. P.; Bulgarelli-Leva, G.; Zvelebil, M. J.; Pirola, L.; Vanhaesebroeck, B.; Waterfield, M. D.; Panayotou, G. Wortmannin Inactivates Phosphoinositide 3-Kinase by Covalent Modification of Lys-802, a Residue Involved in the Phosphate Transfer Reaction. Mol. Cell. Biol. 1996, 16 (4), 1722– 1733, DOI: 10.1128/MCB.16.4.1722[Crossref], [PubMed], [CAS], Google Scholar260https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XhvVags7s%253D&md5=54ebe365b4ad8260555c8b962e03d28bWortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reactionWymann, Matthias P.; Bulgarelli-Leva, Ginette; Zvelebil, Marketa J.; Pirola, Luciano; Vanhaesebroeck, Bart; Waterfield, Michael D.; Panayotou, GeorgeMolecular and Cellular Biology (1996), 16 (4), 1722-33CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Wortmannin at nanomolar concns. is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concns., wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA dependent protein kinase (DNA-PKcs). The authors report here the identification of the site of interaction of wortmannin in the catalytic subunit of PI3-kinase, p110α. At physiol. pH (6.5 to 8) wortmannin reacted specifically with p110α. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances contg. nucleophilic amino acid side chain functions had no effect at the same concns. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant P110α were mapped with anti-wortmannin and anti-p110α peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-59-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-t9-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. The results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiol. processes.
- 261Pettinger, J.; Le Bihan, Y.-V.; Widya, M.; van Montfort, R. L. M.; Jones, K.; Cheeseman, M. D. An Irreversible Inhibitor of HSP72 That Unexpectedly Targets Lysine-56. Angew. Chem., Int. Ed. 2017, 56 (13), 3536– 3540, DOI: 10.1002/anie.201611907[Crossref], [CAS], Google Scholar261https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjtVeru7o%253D&md5=8b2cbd17140e5f0c6430d0164e03280aAn Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine-56Pettinger, Jonathan; Le Bihan, Yann-Vai; Widya, Marcella; van Montfort, Rob L. M.; Jones, Keith; Cheeseman, Matthew D.Angewandte Chemie, International Edition (2017), 56 (13), 3536-3540CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The stress-inducible mol. chaperone, HSP72, is an important therapeutic target in oncol., but inhibiting this protein with small mols. has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine-targeted irreversible inhibitor. Using rational design, we adapted a validated 8-N-benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine-56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chem. probes and drugs.
- 262Altmeyer, M.; Amtmann, E.; Heyl, C.; Marschner, A.; Scheidig, A. J.; Klein, C. D. Beta-Aminoketones as Prodrugs for Selective Irreversible Inhibitors of Type-1 Methionine Aminopeptidases. Bioorg. Med. Chem. Lett. 2014, 24 (22), 5310– 5314, DOI: 10.1016/j.bmcl.2014.09.047[Crossref], [PubMed], [CAS], Google Scholar262https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1eitbjK&md5=e7b2d0e83fb348fdbbb59f622d5fa918Beta-aminoketones as prodrugs for selective irreversible inhibitors of type-1 methionine aminopeptidasesAltmeyer, Markus; Amtmann, Eberhard; Heyl, Carina; Marschner, Aline; Scheidig, Axel J.; Klein, Christian D.Bioorganic & Medicinal Chemistry Letters (2014), 24 (22), 5310-5314CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)We identified and characterized β-aminoketones as prodrugs for irreversible MetAP inhibitors that are selective for the MetAP-1 subtype. β-Aminoketones with certain structural features form α,β-unsatd. ketones under physiol. conditions, which bind covalently and selectively to cysteines in the S1 pocket of MetAP-1. The binding mode was confirmed by X-ray crystallog. and assays with the MetAPs from Escherichia coli, Staphylococcus aureus and both human isoforms. The initially identified tetralone derivs. showed complete selectivity for E. coli MetAP vs. human MetAP-1 and MetAP-2. Rational design of indanone analogs yielded compds. with selectivity for the human type-1 vs. the human type-2 MetAP.
- 263Dahal, U. P.; Gilbert, A. M.; Obach, R. S.; Flanagan, M. E.; Chen, J. M.; Garcia-Irizarry, C.; Starr, J. T.; Schuff, B.; Uccello, D. P.; Young, J. A. Intrinsic Reactivity Profile of Electrophilic Moieties to Guide Covalent Drug Design: N-α-Acetyl-L-Lysine as an Amine Nucleophile. MedChemComm 2016, 7 (5), 864– 872, DOI: 10.1039/C6MD00017G[Crossref], [CAS], Google Scholar263https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XisFyjtrk%253D&md5=08e681ea8dfb353f89d8cdedba685694Intrinsic reactivity profile of electrophilic moieties to guide covalent drug design: N-α-acetyl-L-lysine as an amine nucleophileDahal, Upendra P.; Gilbert, Adam M.; Obach, R. Scott; Flanagan, Mark E.; Chen, Jinshan M.; Garcia-Irizarry, Carmen; Starr, Jeremy T.; Schuff, Brandon; Uccello, Daniel P.; Young, Jennifer A.MedChemComm (2016), 7 (5), 864-872CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)Covalent drugs contain a reactive electrophilic moiety or covalent reactive group (CRG), which forms an irreversible bond between the drug and a biol. target. Consequently, the intrinsic reactivity of the CRG is an important consideration in the design of irreversible inhibitors. Although reactivity assessments of CRGs with sulfur nucleophiles, such as glutathione and cysteine have been reported, reactivity of these moieties with amine-contg. nucleophiles is not well described. In this study, intrinsic reactivities were detd. for a series of electrophiles (acrylamides, nitriles, cyanamides, sulfones, and sulfonamides) using N-α-acetyl-L-lysine as a model amine-based nucleophile and compared with results using glutathione (GSH). Since the ε-amine of N-α-acetyl-L-lysine is protonated at neutral pH, reactions were carried out at pH 10.2. In addn. to reporting rate data for reactions of CRGs with N-α-acetyl-L-lysine, elements of selectivity relative to thiol-contg. nucleophiles are also be discussed.
- 264Anscombe, E.; Meschini, E.; Mora-Vidal, R.; Martin, M. P.; Staunton, D.; Geitmann, M.; Danielson, U. H.; Stanley, W. A.; Wang, L. Z.; Reuillon, T.; Golding, B. T.; Cano, C.; Newell, D. R.; Noble, M. E. M.; Wedge, S. R.; Endicott, J. A.; Griffin, R. J. Identification and Characterization of an Irreversible Inhibitor of CDK2. Chem. Biol. 2015, 22 (9), 1159– 1164, DOI: 10.1016/j.chembiol.2015.07.018[Crossref], [PubMed], [CAS], Google Scholar264https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVajsL7M&md5=e6a29b14eb31abd659a48511bb11ed98Identification and Characterization of an Irreversible Inhibitor of CDK2Anscombe, Elizabeth; Meschini, Elisa; Mora-Vidal, Regina; Martin, Mathew P.; Staunton, David; Geitmann, Matthis; Danielson, U. Helena; Stanley, Will A.; Wang, Lan Z.; Reuillon, Tristan; Golding, Bernard T.; Cano, Celine; Newell, David R.; Noble, Martin E. M.; Wedge, Stephen R.; Endicott, Jane A.; Griffin, Roger J.Chemistry & Biology (Oxford, United Kingdom) (2015), 22 (9), 1159-1164CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclohexylmethoxy)-9H-purin-2-yl)amino)benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl)phenyl)-9H-purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compds.
- 265Narayanan, A.; Jones, L. H. Sulfonyl Fluorides as Privileged Warheads in Chemical Biology. Chem. Sci. 2015, 6 (5), 2650– 2659, DOI: 10.1039/C5SC00408J[Crossref], [PubMed], [CAS], Google Scholar265https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksVamurc%253D&md5=0009feb4068e912b4784fb8411cdb702Sulfonyl fluorides as privileged warheads in chemical biologyNarayanan, Arjun; Jones, Lyn H.Chemical Science (2015), 6 (5), 2650-2659CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Sulfonyl fluoride electrophiles have found significant utility as reactive probes in chem. biol. and mol. pharmacol. As warheads they possess the right balance of biocompatibility (including aq. stability) and protein reactivity. Their functionality is privileged in this regard as they are known to modify not only reactive serines (resulting in their common use as protease inhibitors), but also context-specific threonine, lysine, tyrosine, cysteine and histidine residues. This review describes the application of sulfonyl fluoride probes across various areas of research and explores new approaches that could further enhance the chem. biol. toolkit. We believe that sulfonyl fluoride probes will find greater utility in areas such as covalent enzyme inhibition, target identification and validation, and the mapping of enzyme binding sites, substrates and protein-protein interactions.
- 266Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B. Sulfur(VI) Fluoride Exchange (SuFEx): Another Good Reaction for Click Chemistry. Angew. Chem., Int. Ed. 2014, 53 (36), 9430– 9448, DOI: 10.1002/anie.201309399[Crossref], [CAS], Google Scholar266https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtleisbzJ&md5=78e251c607d42f2506a9420f2f2f9835Sulfur(VI) Fluoride Exchange (SuFEx): Another Good Reaction for Click ChemistryDong, Jiajia; Krasnova, Larissa; Finn, M. G.; Sharpless, K. BarryAngewandte Chemie, International Edition (2014), 53 (36), 9430-9448CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Aryl sulfonyl chlorides (e.g. Ts-Cl) are beloved of org. chemists as the most commonly used SVI electrophiles, and the parent sulfuryl chloride, O2SVICl2, also was relied on to create sulfates and sulfamides. However, the desired halide substitution event is often defeated by destruction of the sulfur electrophile because the SVI-Cl bond is exceedingly sensitive to reductive collapse yielding SIV species and Cl-. Fortunately, the use of sulfur(VI) fluorides (e.g., R-SO2-F and SO2F2) leaves only the substitution pathway open. As with most of click chem., many essential features of sulfur(VI) fluoride reactivity were discovered long ago in Germany. Surprisingly, this extraordinary work faded from view rather abruptly in the mid-20th century. Here the authors seek to revive it, along with John Hyatt's unnoticed 1979 full paper exposition on CH2=CH-SO2-F, the most perfect Michael acceptor ever found. To this history the authors add several new observations, including that the otherwise very stable gas SO2F2 has excellent reactivity under the right circumstances. Also proton or silicon centers can activate the exchange of S-F bonds for S-O bonds to make functional products, and the sulfate connector is surprisingly stable toward hydrolysis. Applications of this controllable ligation chem. to small mols., polymers, and biomols. are discussed.
- 267Baker, B. R. Irreversible Enzyme Inhibitors. CXLIX. Tissue-Specific Irreversible Inhibitors of Dihydrofolic Reductase. Acc. Chem. Res. 1969, 2 (5), 129– 136, DOI: 10.1021/ar50017a001[ACS Full Text], [CAS], Google Scholar267https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF1MXksVKjsL4%253D&md5=bc7effcdf83f4fae3ec4a90f971ea4f0Irreversible enzyme inhibitors. CXLIX. Tissue-specific irreversible inhibitors of dihydrofolic reductaseBaker, Bernard RandallAccounts of Chemical Research (1969), 2 (5), 129-36CODEN: ACHRE4; ISSN:0001-4842.Inhibitors that partly complex in the active site and partly complex in the hydrophobic bonding region can be constructed to show huge differences in binding between mammalian enzymes on one hand and protozoal enzymes on the other. This is of chemotherapeutic utility. 17 references.
- 268Chen, W.; Dong, J.; Plate, L.; Mortenson, D. E.; Brighty, G. J.; Li, S.; Liu, Y.; Galmozzi, A.; Lee, P. S.; Hulce, J. J.; Cravatt, B. F.; Saez, E.; Powers, E. T.; Wilson, I. A.; Sharpless, K. B.; Kelly, J. W. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue. J. Am. Chem. Soc. 2016, 138 (23), 7353– 7364, DOI: 10.1021/jacs.6b02960[ACS Full Text], [CAS], Google Scholar268https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XotVylu7Y%253D&md5=d946cba154b07a712ceae9e8136f8480Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr ResidueChen, Wentao; Dong, Jiajia; Plate, Lars; Mortenson, David E.; Brighty, Gabriel J.; Li, Suhua; Liu, Yu; Galmozzi, Andrea; Lee, Peter S.; Hulce, Jonathan J.; Cravatt, Benjamin F.; Saez, Enrique; Powers, Evan T.; Wilson, Ian A.; Sharpless, K. Barry; Kelly, Jeffery W.Journal of the American Chemical Society (2016), 138 (23), 7353-7364CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-mol. ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis expts., provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function.
- 269Mortenson, D. E.; Brighty, G. J.; Plate, L.; Bare, G.; Chen, W.; Li, S.; Wang, H.; Cravatt, B. F.; Forli, S.; Powers, E. T.; Sharpless, K. B.; Wilson, I. A.; Kelly, J. W. “Inverse Drug Discovery” Strategy To Identify Proteins That Are Targeted by Latent Electrophiles As Exemplified by Aryl Fluorosulfates. J. Am. Chem. Soc. 2018, 140 (1), 200– 210, DOI: 10.1021/jacs.7b08366[ACS Full Text], [CAS], Google Scholar269https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitVShtLbF&md5=cfeb5f1ae67fe89340fffaa4cf5e46a8"Inverse Drug Discovery" Strategy To Identify Proteins That Are Targeted by Latent Electrophiles As Exemplified by Aryl FluorosulfatesMortenson, David E.; Brighty, Gabriel J.; Plate, Lars; Bare, Grant; Chen, Wentao; Li, Suhua; Wang, Hua; Cravatt, Benjamin F.; Forli, Stefano; Powers, Evan T.; Sharpless, K. Barry; Wilson, Ian A.; Kelly, Jeffery W.Journal of the American Chemical Society (2018), 140 (1), 200-210CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Drug candidates are generally discovered using biochem. screens employing an isolated target protein or by utilizing cell-based phenotypic assays. Both noncovalent and covalent hits emerge from such endeavors. Herein, we exemplify an "Inverse Drug Discovery" strategy in which org. compds. of intermediate complexity harboring weak, but activatable, electrophiles are matched with the protein(s) they react with in cells or cell lysate. An alkyne substructure in each candidate small mol. enables affinity chromatog.-mass spectrometry, which produces a list of proteins that each distinct compd. reacts with. A notable feature of this approach is that it is agnostic with respect to the cellular proteins targeted. To illustrate this strategy, we employed aryl fluorosulfates, an underexplored class of sulfur(VI) halides, that are generally unreactive unless activated by protein binding. Reversible aryl fluorosulfate binding, correct juxtaposition of protein side chain functional groups, and transition-state stabilization of the S(VI) exchange reaction all seem to be crit. for conjugate formation. The aryl fluorosulfates studied thus far exhibit chemoselective reactivity toward Lys and, particularly, Tyr side chains, and can be used to target nonenzymes (e.g., a hormone carrier or a small-mol. carrier protein) as well as enzymes. The "Inverse Drug Discovery" strategy should be particularly attractive as a means to explore latent electrophiles not typically used in medicinal chem. efforts, until one reacts with a protein target of exceptional interest. Structure-activity data can then be used to enhance the selectivity of conjugate formation or the covalent probe can be used as a competitor to develop noncovalent drug candidates. Here we use the "Inverse Drug Discovery" platform to identify and validate covalent ligands for 11 different human proteins. In the case of one of these proteins, we have identified and validated a small-mol. probe for the first time.
- 270James, G. T. Inactivation of the Protease Inhibitor Phenylmethylsulfonyl Fluoride in Buffers. Anal. Biochem. 1978, 86 (2), 574– 579, DOI: 10.1016/0003-2697(78)90784-4[Crossref], [PubMed], [CAS], Google Scholar270https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXktF2jtr8%253D&md5=71a4087d48cfdee57eb4d70c9e8b7d2fInactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffersJames, Gordon T.Analytical Biochemistry (1978), 86 (2), 574-9CODEN: ANBCA2; ISSN:0003-2697.Aq. prepns. of phenylmethylsulfonyl fluoride (PMSF) are inactive toward proteases unless promptly brought into contact with protease. Inactivation of PMSF increased with increased pH and temp. Halflives of the inhibitor at 25° were ∼110, 55, and 35 min at pH 7.0, 7.5, and 8.0, resp. At pH 8,100 μM PMSF was almost completely inactivated within 1 h at 25° or within 22 h at 4°. Stock solns. of PMSF in 100% isopropanol were stable at 25° for many months. Reactivation of PMSF-inhibited chymotrypsin did not occur within 1 wk at 25° at pH 7.0.
- 271Lively, M. O.; Powers, J. C. Specificity and Reactivity of Human Granulocyte Elastase and Cathepsin G, Porcine Pancreatic Elastase, Bovine Chymotrypsin and Trypsin toward Inhibition with Sulfonyl Flourides. Biochim. Biophys. Acta BBA - Enzymol. 1978, 525 (1), 171– 179, DOI: 10.1016/0005-2744(78)90211-5
- 272Genov, N. C.; Shopova, M.; Boteva, R.; Ricchelli, F.; Jori, G. Intramolecular Distances between Tryptophan Residues and the Active-Site Serine Residue in Alkaline Bacterial Proteinases as Measured by Fluorescence Energy-Transfer Studies. Biochem. J. 1983, 215 (2), 413– 416, DOI: 10.1042/bj2150413[Crossref], [PubMed], [CAS], Google Scholar272https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXmtVykurk%253D&md5=3db650c76b172dfe1e3f21c2f125361dIntramolecular distances between tryptophan residues and the active-site serine residue in alkaline bacterial proteinases as measured by fluorescence energy-transfer studiesGenov, N.; Shopova, M.; Boteva, R.; Ricchelli, F.; Jori, G.Biochemical Journal (1983), 215 (2), 413-16CODEN: BIJOAK; ISSN:0264-6021.Singlet-singlet energy transfer from the tryptophan residues to an active-site-serine-bound 5-dimethylaminonaphthalene-1-sulfonyl group was investigated in 4 subtilisins. The transfer distance for subtilisin Novo and mesentericopeptidase are 1.93 and 1.81 nm, resp. The positions of the indole groups in the 3-dimensional structures of 2 pairs of proteinases, namely subtilisin Novo and mesentericopeptidase on the one hand and subtilisins Carlsberg and DY on the other, are essentially identical.
- 273Esch, F. S.; Allison, W. S. Identification of a Tyrosine Residue at a Nucleotide Binding Site in the Beta Subunit of the Mitochondrial ATPase with P-Fluorosulfonyl[14C]-Benzoyl-5′-Adenosine. J. Biol. Chem. 1978, 253, 6100– 6106[PubMed], [CAS], Google Scholar273https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXlslGlurc%253D&md5=a04ea874870c890bca5148857ebe364eIdentification of a tyrosine residue at a nucleotide binding site in the β subunit of the mitochondrial ATPase with p-fluorosulfonyl[14C]-benzoyl-5'-adenosineEsch, Fred S.; Allison, William S.Journal of Biological Chemistry (1978), 253 (17), 6100-6CODEN: JBCHA3; ISSN:0021-9258.Bovine heart mitochondrial F1-ATPase is irreversibly inactivated by the adenine nucleotide analog, p-fluorosulfonylbenzoyl-5'-adenosine (I). This inactivation is partly prevented by the presence of bound adenine nucleotides. Inactivations of the ATPase with I-14C were most efficiently accomplished with the nucleotide-free enzyme at pH 7.0, in a buffer contg. 20% glycerol. Under these conditions, 4.2 g-atoms of 14C are incorporated/350,000 g enzyme when the ATPase is inactivated by 90% by its reaction with 2 mM I-14C. Isolation of the component polypeptide chains of the labeled ATPase showed that all of the radioactivity was assocd. with the 2 largest subunits. The isolated α subunit contained 0.45 g-atom 14C/mol and the isolated β subunit contained 0.88 g atom 14C/mol. Hence, the inactivation can be correlated with the incorporation of 14C into the β subunit. This suggests that the hydrolytic site of the enzyme resides on this subunit. The majority of the radioactivity in a tryptic digest of labeled β subunit is contained in a tryptic peptide that has the following amino acid sequence: Ile-Met-Asp-Pro-Asn-Ile-Val-Gly-Ser-Glu-His-*Tyr-Asp-Val-Ala-Arg, where *Tyr is the radioactive deriv. of the tyrosine residue that was sulfonylated during the inactivation.
- 274Jörg, M.; Glukhova, A.; Abdul-Ridha, A.; Vecchio, E. A.; Nguyen, A. T. N.; Sexton, P. M.; White, P. J.; May, L. T.; Christopoulos, A.; Scammells, P. J. Novel Irreversible Agonists Acting at the A1 Adenosine Receptor. J. Med. Chem. 2016, 59 (24), 11182– 11194, DOI: 10.1021/acs.jmedchem.6b01561[ACS Full Text], [CAS], Google Scholar274https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFynt77F&md5=6157d0f8b0b980dedf4462ddd263a807Novel Irreversible Agonists Acting at the A1 Adenosine ReceptorJorg, Manuela; Glukhova, Alisa; Abdul-Ridha, Alaa; Vecchio, Elizabeth A.; Nguyen, Anh T. N.; Sexton, Patrick M.; White, Paul J.; May, Lauren T.; Christopoulos, Arthur; Scammells, Peter J.Journal of Medicinal Chemistry (2016), 59 (24), 11182-11194CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The A1 adenosine receptor (A1AR) is an important G protein-coupled receptor that regulates a range of physiol. functions. Herein we report the discovery of novel irreversible agonists acting at the A1AR, which have the potential to serve as useful research tools for studying receptor structure and function. A series of novel adenosine derivs. bearing electrophilic substituents was synthesized, and four compds., 8b, 15a, 15b, and 15d, were shown to possess similar potency and efficacy to the ref. high efficacy agonist, NECA, in an assay of ERK1/2 phosphorylation assay. Insensitivity to antagonist addn. in a real-time, label-free, xCELLigence assay was subsequently used to identify compds. that likely mediated their agonism through an irreversible interaction with the A1AR. Of these compds., 15b and 15d were more directly validated as irreversible agonists of the A1AR using membrane-based [3H]DPCPX and [35S]GTPγS binding expts.
- 275Yang, X.; Dong, G.; Michiels, T. J. M.; Lenselink, E. B.; Heitman, L.; Louvel, J.; IJzerman, A. P. A Covalent Antagonist for the Human Adenosine A2A Receptor. Purinergic Signalling 2017, 13 (2), 191– 201, DOI: 10.1007/s11302-016-9549-9[Crossref], [PubMed], [CAS], Google Scholar275https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVSrtLrN&md5=7ce9f2fd7be76d83997cd3aac8eb91c1A covalent antagonist for the human adenosine A2A receptorYang, Xue; Dong, Guo; Michiels, Thomas J. M.; Lenselink, Eelke B.; Heitman, Laura; Louvel, Julien; IJzerman, Ad P.Purinergic Signalling (2017), 13 (2), 191-201CODEN: PSUIA9; ISSN:1573-9538. (Springer)The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallog. with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template mol. served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out expts. was set up to investigate the efficacy of covalent binding of LUF7445. All these expts. led us to conclude LUF7445 is a valuable mol. tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.
- 276Beauglehole, A. R.; Baker, S. P.; Scammells, P. J. Fluorosulfonyl-Substituted Xanthines as Selective Irreversible Antagonists for the A1-Adenosine Receptor. J. Med. Chem. 2000, 43 (26), 4973– 4980, DOI: 10.1021/jm000181f[ACS Full Text], [CAS], Google Scholar276https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXotFymurw%253D&md5=cf1fdfea7ede2f1f5874d0bd82ff9b38Fluorosulfonyl-Substituted Xanthines as Selective Irreversible Antagonists for the A1-Adenosine ReceptorBeauglehole, Anthony R.; Baker, Stephen P.; Scammells, Peter J.Journal of Medicinal Chemistry (2000), 43 (26), 4973-4980CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)FSCPX (1) has been reported to be a potent, selective, and irreversible antagonist for the A1-adenosine receptor (AR). To obtain an irreversible A1AR antagonist with potentially better stability and to further elucidate the effects of linker structure on the pharmacol. characteristics, several new analogs were targeted in which the labile ester linkage of 1 was replaced by more stable functionalities. In particular, alkyl and amide linkers between the xanthine pharmacophore and the reactive 4-fluorosulfonylphenyl group were explored. The data showed that the chem. compn. of the linker affects the affinity and apparent irreversible binding to the A1AR. Overall, compd. (I) appeared to have the most advantageous characteristics as a potential irreversible ligand for the A1AR. These include relatively high affinity for the A1AR as compared to the A2AAR, concn.-dependent and selective apparent irreversible binding to the A1AR, and ease of removal of unbound ligand from biol. membranes. These properties indicate that I has the potential to be a useful tool for further study of the structure and function of the A1AR.
- 277Glukhova, A.; Thal, D. M.; Nguyen, A. T.; Vecchio, E. A.; Jörg, M.; Scammells, P. J.; May, L. T.; Sexton, P. M.; Christopoulos, A. Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity. Cell 2017, 168, 867– 877, DOI: 10.1016/j.cell.2017.01.042[Crossref], [PubMed], [CAS], Google Scholar277https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjvVWrsrg%253D&md5=a5ff676cfedd6e11a899f65555a1ba62Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype SelectivityGlukhova, Alisa; Thal, David M.; Nguyen, Anh T.; Vecchio, Elizabeth A.; Jorg, Manuela; Scammells, Peter J.; May, Lauren T.; Sexton, Patrick M.; Christopoulos, ArthurCell (Cambridge, MA, United States) (2017), 168 (5), 867-877.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The adenosine A1 receptor (A1-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We detd. a 3.2 Å crystal structure of the A1-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A2A receptor (A2A-AR) structure. Mutational and computational anal. of A1-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a mol. basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.
- 278Kitz, R.; Wilson, I. B. Esters of Methanesulfonic Acid as Irreversible Inhibitors of Acetylcholinesterase. J. Biol. Chem. 1962, 237, 3245– 3249[PubMed], [CAS], Google Scholar278https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF38Xks1Cmsrg%253D&md5=f3993cf79ebb66299fada0ad04b15860Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesteraseKitz, R.; Wilson, Irwin B.Journal of Biological Chemistry (1962), 237 (), 3245-49CODEN: JBCHA3; ISSN:0021-9258.Nine esters (anhydrides) of methanesulfonic acid, analogs to well known carbamate (e.g. neostigmine) and phosphate (e.g. paraoxon) inhibitors, were examd. as possible irreversible inhibitors of acetylcholinesterase. Five were active. Of these, 4 formed initial reversible complexes. The progressive development of irreversible inhibition was slowed by reversible inhibitors, indicating that the active site is involved. The inhibited enzyme is not reactivated by water or by hydroxylamine, hydrazine, acetate, or pyridine-2-aldoxime methiodide. It is reactivated by pyridine-3-aldoxime methiodide. On the basis of the evidence presented, it may be concluded safely that the active methanesulfonates form methanesulfonyl enzyme derivs. and, therefore, belong to the class of acid-transferring or oxydiaphoric inhibitors.
- 279Moss, D. E.; Berlanga, P.; Hagan, M. M.; Sandoval, H.; Ishida, C. Methanesulfonyl Fluoride (MSF): A Double-Blind, Placebo-Controlled Study of Safety and Efficacy in the Treatment of Senile Dementia of the Alzheimer Type. Alzheimer Dis. Assoc. Disord. 1999, 13 (1), 20, DOI: 10.1097/00002093-199903000-00003[Crossref], [PubMed], [CAS], Google Scholar279https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXitVeqtL4%253D&md5=64f9fda8b3410f6d17ce7ba4704785cdMethanesulfonyl fluoride (MSF): a double-blind, placebo-controlled study of safety and efficacy in the treatment of senile dementia of the Alzheimer typeMoss, D. E.; Berlanga, P.; Hagan, M. M.; Sandoval, H.; Ishida, C.Alzheimer Disease and Associated Disorders (1999), 13 (1), 20-25CODEN: ADADE2; ISSN:0893-0341. (Lippincott Williams & Wilkins)The purpose of the present study was to evaluate methanesulfonyl fluoride (MSF), a very long-acting CNS-selective acetylcholinesterase (AChE) inhibitor, as a palliative treatment for senile dementia of the Alzheimer type (SDAT). In expt. I, MSF (0.03-0.18 mg/kg) was administered orally to 10 normal volunteers to measure toxicity and establish dose/response function in erythrocyte AChE. MSF produced a dose-response function of %inhibition = (40)(Log10[MSF mg/kg] + 51.7) with no toxicity at these doses. Expt. II was a 16-wk double-blind, placebo-controlled study of the safety and efficacy of MSF in doses of up to 0.18 mg/kg given three times per wk in 5 men and 10 women (60-82 yr), with Mini-Mental State Examn. (MMSE) scores of 9-24, who had SDAT. MSF produced a mean of 89.5% inhibition of erythrocyte AChE in patients and improved cognitive performance as measured by the MMSE, Alzheimer Disease Assessment Scale-Cognitive Subscale (ADAS-COG), Global Deterioration Scale, and the Clin. Interview Based Impression of Change (CIBIC). Most of the improvement on the ADAS-COG was maintained 8 wk after ending MSF. No patients left the study because of drug-related adverse events and there were no toxic effects. MSF may be a safe and effective palliative treatment for SDAT and further clin. trials in larger groups of patients are warranted.
- 280Moss, D. E.; Fariello, R. G.; Sahlmann, J.; Sumaya, I.; Pericle, F.; Braglia, E. A Randomized Phase I Study of Methanesulfonyl Fluoride, an Irreversible Cholinesterase Inhibitor, for the Treatment of Alzheimer’s Disease. Br. J. Clin. Pharmacol. 2013, 75 (5), 1231– 1239, DOI: 10.1111/bcp.12018[Crossref], [PubMed], [CAS], Google Scholar280https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXps1CqsLo%253D&md5=ef0e3ba992302e53d74ade7647d8554cA randomized phase I study of methanesulfonyl fluoride, an irreversible cholinesterase inhibitor, for the treatment of Alzheimer's diseaseMoss, Donald E.; Fariello, Ruggero G.; Sahlmann, Jorg; Sumaya, Isabel; Pericle, Federica; Braglia, EnricoBritish Journal of Clinical Pharmacology (2013), 75 (5), 1231-1239CODEN: BCPHBM; ISSN:1365-2125. (Wiley-Blackwell)AIMS: To ascertain the tolerability profile of single and repeated oral doses of methanesulfonyl fluoride (MSF, SNX-001) in healthy aged subjects, and to det. the degree of erythrocyte acetylcholinesterase (AChE) inhibition induced by MSF after single and repeated oral doses. METHODS: To calc. properly the kinetics and the duration of AChE inhibition, the effects of MSF were also studied in rodents. These expts. suggested that MSF administered three times per wk should provide safe and efficacious AChE inhibition. In a randomized placebo-controlled phase I study, 3.6 mg, 7.2 mg or 10.8 mg MSF were then orally administered to 27 consenting healthy volunteers (aged 50 to 72 years). After a single dose phase and a 1 wk wash-out period, the subjects received the same doses three times per wk for 2 wk. RESULTS: Twenty-two out of the 27 subjects completed the study. Four patients withdrew due to adverse events (AEs) and one for non-compliance. Erythrocyte AChE was inhibited by a total of 33%, 46%, and 62% after 2 wk of 3.6 mg, 7.2 mg and 10.8 mg MSF, resp. No serious AEs occurred. The most frequent AEs were headache (27%), nausea (11%) and diarrhea (8%). CONCLUSIONS: MSF proved to be well tolerated even with repeated oral dosing. It is estd. that MSF provided a degree of AChE inhibition that should effectively enhance memory. This mol. deserves to be tested for efficacy in a pilot randomized controlled study in patients with Alzheimer's disease.
- 281Jones, L. H. Reactive Chemical Probes: Beyond the Kinase Cysteinome. Angew. Chem., Int. Ed. 2018, 57 (30), 9220– 9223, DOI: 10.1002/anie.201802693[Crossref], [CAS], Google Scholar281https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFaltLbF&md5=3aa3b629e71ea55d7627c2a94da895efReactive Chemical Probes: Beyond the Kinase CysteinomeJones, Lyn H.Angewandte Chemie, International Edition (2018), 57 (30), 9220-9223CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The reaction of small-mol. chem. probes with proteins has been harnessed to develop covalent inhibitor drugs and protein-profiling technologies. This Essay discusses some of the recent enhancements to the chem. biol. toolkit that are enabling the study of previously uncharted areas of chemoproteomic space. An anal. of the kinome is used to illustrate the potential for these approaches enable the pursuit of new targets using reactive chem. probes.
- 282Mukherjee, H.; Debreczeni, J.; Breed, J.; Tentarelli, S.; Aquila, B.; Dowling, J. E.; Whitty, A.; Grimster, N. P. A Study of the Reactivity of S(VI)–F Containing Warheads with Nucleophilic Amino-Acid Side Chains under Physiological Conditions. Org. Biomol. Chem. 2017, 15 (45), 9685– 9695, DOI: 10.1039/C7OB02028G[Crossref], [PubMed], [CAS], Google Scholar282https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslSgsbnI&md5=cf00897a4b0b184e2d68df498ec8cbb7A study of the reactivity of S(VI)-F containing warheads with nucleophilic amino-acid side chains under physiological conditionsMukherjee, H.; Debreczeni, J.; Breed, J.; Tentarelli, S.; Aquila, B.; Dowling, J. E.; Whitty, A.; Grimster, N. P.Organic & Biomolecular Chemistry (2017), 15 (45), 9685-9695CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Sulfonyl fluorides (SFs) have recently emerged as a promising warhead for the targeted covalent modification of proteins. Despite numerous examples of the successful deployment of SFs as covalent probe compds., a detailed exploration of the factors influencing the stability and reactivity of SFs has not yet appeared. In this work we present an extensive study on the influence of steric and electronic factors on the reactivity and stability of the SF and related SVI-F groups. While SFs react rapidly with N-acetylcysteine, the resulting adducts were found to be unstable, rendering SFs inappropriate for the durable covalent inhibition of cysteine residues. In contrast, SFs afforded stable adducts with both N-acetyltyrosine and N-acetyllysine; furthermore, we show that the reactivity of arylsulfonyl fluorides towards these nucleophilic amino acids can be predictably modulated by adjusting the electronic properties of the warhead. These trends were largely conserved when the covalent reaction occurred within a protein binding pocket. We have also obtained a crystal structure depicting covalent modification of the catalytic lysine of a tyrosine kinase (FGFR1) by the ATP analog 5'-O-3-((fluorosulfonyl)benzoyl)adenosine (m-FSBA). Highly reactive warheads were demonstrated to be unstable with respect to hydrolysis in buffered aq. solns., indicating that warhead reactivity must be carefully tuned to provide optimal rates of protein modification. Our results demonstrate that the reactivity of SFs complements that of more commonly studied acrylamides, and we hope that this work spurs the rational design of novel SF-contg. covalent probe compds. and inhibitors, particularly in cases where a suitably positioned cysteine residue is not present.
- 283Lundblad, R. L. Chemical Reagents for Protein Modification, 4th ed.; CRC Press: Boca Raton, 2017.
- 284Chinthakindi, P. K.; Arvidsson, P. I. Sulfonyl Fluorides (SFs): More Than Click Reagents?. Eur. J. Org. Chem. 2018, 2018 (27–28), 3648– 3666, DOI: 10.1002/ejoc.201800464[Crossref], [CAS], Google Scholar284https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1yktL%252FE&md5=819292301bdb81cab0f12f4c535d8078Sulfonyl Fluorides (SFs): More Than Click Reagents?Chinthakindi, Praveen K.; Arvidsson, Per I.European Journal of Organic Chemistry (2018), 2018 (27-28), 3648-3666CODEN: EJOCFK; ISSN:1099-0690. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Sulfonyl fluoride (SF)-contg. substances are currently attracting enormous attention among practitioners of both chem. biol. and synthetic org. chem. The groups of Jones and Liskamp have demonstrated the potential of sulfonyl fluorides as selective covalent inhibitors in studies related to drug discovery and chem. biol., resp., in the last few years. The Sharpless group has extended the repertoire of "click-reactions" to those involving sulfonyl fluorides, i.e., sulfur-fluoride exchange (SuFEx), a development that quickly triggered the interest in this functional group in the community of synthetic org. chemists. In this microreview, we aim to give an account of the synthetic chem. surrounding sulfonyl fluoride contg. substances from a historical perspective to present day developments.
- 285Wang, N.; Yang, B.; Fu, C.; Zhu, H.; Zheng, F.; Kobayashi, T.; Liu, J.; Li, S.; Ma, C.; Wang, P. G.; Wang, Q.; Wang, L. Genetically Encoding Fluorosulfate-l-Tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo. J. Am. Chem. Soc. 2018, 140 (15), 4995– 4999, DOI: 10.1021/jacs.8b01087[ACS Full Text], [CAS], Google Scholar285https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmslGqs78%253D&md5=30583248ed49b2499ef4fd1481ac5adfGenetically Encoding Fluorosulfate-L-tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in VivoWang, Nanxi; Yang, Bing; Fu, Caiyun; Zhu, He; Zheng, Feng; Kobayashi, Tomonori; Liu, Jun; Li, Shanshan; Ma, Cheng; Wang, Peng G.; Wang, Qian; Wang, LeiJournal of the American Chemical Society (2018), 140 (15), 4995-4999CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Introducing new chem. reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering in vivo. Latent bioreactive unnatural amino acids (Uaas) can be incorporated into proteins to react with target natural amino acid residues via proximity-enabled reactivity. To expand the diversity of proteins amenable to such reactivity in vivo, a chem. functionality that is biocompatible and able to react with multiple natural residues under physiol. conditions is highly desirable. Here the authors report the genetic encoding of fluorosulfate-L-tyrosine (FSY), the first latent bioreactive Uaa that undergoes sulfur-fluoride exchange (SuFEx) on proteins in vivo. FSY was found nontoxic to Escherichia coli and mammalian cells; after being incorporated into proteins, it selectively reacted with proximal lysine, histidine, and tyrosine via SuFEx, generating covalent intraprotein bridge and interprotein crosslink of interacting proteins directly in living cells. The proximity-activatable reactivity, multitargeting ability, and excellent biocompatibility of FSY will be invaluable for covalent manipulation of proteins in vivo. Moreover, genetically encoded FSY hereby empowers general proteins with the next generation of click chem., SuFEx, which will afford broad utilities in chem. biol., drug discovery, and biotherapeutics.
- 286Zhao, Q.; Ouyang, X.; Wan, X.; Gajiwala, K. S.; Kath, J. C.; Jones, L. H.; Burlingame, A. L.; Taunton, J. Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes. J. Am. Chem. Soc. 2017, 139 (2), 680– 685, DOI: 10.1021/jacs.6b08536[ACS Full Text], [CAS], Google Scholar286https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslWisA%253D%253D&md5=3f643a1b32c83fa05932f42b54e96e47Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride ProbesZhao, Qian; Ouyang, Xiaohu; Wan, Xiaobo; Gajiwala, Ketan S.; Kath, John C.; Jones, Lyn H.; Burlingame, Alma L.; Taunton, JackJournal of the American Chemical Society (2017), 139 (2), 680-685CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallog. and mass spectrometry (MS) confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concns. of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clin. relevant concns., highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications.
- 287Becher, I.; Savitski, M. M.; Savitski, M. F.; Hopf, C.; Bantscheff, M.; Drewes, G. Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP. ACS Chem. Biol. 2013, 8 (3), 599– 607, DOI: 10.1021/cb3005879[ACS Full Text], [CAS], Google Scholar287https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhslyjsL7P&md5=a9c8ac0cfbdcda3f20f2d0d91422374fAffinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTPBecher, Isabelle; Savitski, Mikhail M.; Savitski, Maria Faelth; Hopf, Carsten; Bantscheff, Marcus; Drewes, GerardACS Chemical Biology (2013), 8 (3), 599-607CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Most kinase inhibitor drugs target the binding site of the nucleotide cosubstrate ATP. The high intracellular concn. of ATP can strongly affect inhibitor potency and selectivity depending on the affinity of the target kinase for ATP. Here we used a defined chemoproteomics system based on competition-binding assays in cell exts. from Jurkat and SK-MEL-28 cells with immobilized ATP mimetics (kinobeads). This system enabled us to assess the affinities of more than 200 kinases for the cellular nucleotide cofactors ATP, ADP, and GTP and the effects of the divalent metal ions Mg2+ and Mn2+. The affinity values detd. in this system were largely consistent across the two cell lines, indicating no major dependence on kinase expression levels. Kinase-ATP affinities range from low micromolar to millimolar, which has profound consequences for the prediction of cellular effects from inhibitor selectivity profiles. Only a small no. of kinases including CK2, MEK, and BRAF exhibited affinity for GTP. This extensive and consistent data set of kinase-nucleotide affinities, detd. for native enzymes under defined exptl. conditions, will represent a useful resource for kinase drug discovery.
- 288Baranczak, A.; Liu, Y.; Connelly, S.; Du, W.-G. H.; Greiner, E. R.; Genereux, J. C.; Wiseman, R. L.; Eisele, Y. S.; Bradbury, N. C.; Dong, J.; Noodleman, L.; Sharpless, K. B.; Wilson, I. A.; Encalada, S. E.; Kelly, J. W. A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis Elegans. J. Am. Chem. Soc. 2015, 137 (23), 7404– 7414, DOI: 10.1021/jacs.5b03042[ACS Full Text], [CAS], Google Scholar288https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVans7bN&md5=ba307289df4c28c469905a8250d11126A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis elegansBaranczak, Aleksandra; Liu, Yu; Connelly, Stephen; Du, Wen-Ge Han; Greiner, Erin R.; Genereux, Joseph C.; Wiseman, R. Luke; Eisele, Yvonne S.; Bradbury, Nadine C.; Dong, Jiajia; Noodleman, Louis; Sharpless, K. Barry; Wilson, Ian A.; Encalada, Sandra E.; Kelly, Jeffery W.Journal of the American Chemical Society (2015), 137 (23), 7404-7414CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Fluorogenic probes, due to their often greater spatial and temporal sensitivity in comparison to permanently fluorescent small mols., represent powerful tools to study protein localization and function in the context of living systems. Herein, we report fluorogenic probe 4, a 1,3,4-oxadiazole designed to bind selectively to transthyretin (TTR). Probe 4 comprises a fluorosulfate group not previously used in an environment-sensitive fluorophore. The fluorosulfate functional group does not react covalently with TTR on the time scale required for cellular imaging, but does red shift the emission max. of probe 4 in comparison to its nonfluorosulfated analog. We demonstrate that probe 4 is dark in aq. buffers, whereas the TTR·4 complex exhibits a fluorescence emission max. at 481 nm. The addn. of probe 4 to living HEK293T cells allows efficient binding to and imaging of exogenous TTR within intracellular organelles, including the mitochondria and the endoplasmic reticulum. Furthermore, live Caenorhabditis elegans expressing human TTR transgenically and treated with probe 4 display TTR·4 fluorescence in macrophage-like coelomocytes. An analog of fluorosulfate probe 4 does react selectively with TTR without labeling the remainder of the cellular proteome. Studies on this analog suggest that certain aryl fluorosulfates, due to their cell and organelle permeability and activatable reactivity, could be considered for the development of protein-selective covalent probes.
- 289Grimster, N. P.; Connelly, S.; Baranczak, A.; Dong, J.; Krasnova, L. B.; Sharpless, K. B.; Powers, E. T.; Wilson, I. A.; Kelly, J. W. Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis While Affording a Fluorescent Conjugate. J. Am. Chem. Soc. 2013, 135 (15), 5656– 5668, DOI: 10.1021/ja311729d[ACS Full Text], [CAS], Google Scholar289https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFejsrs%253D&md5=d019c851c9fcdc108a8de6af617e1b5bAromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis while Affording a Fluorescent ConjugateGrimster, Neil P.; Connelly, Stephen; Baranczak, Aleksandra; Dong, Jiajia; Krasnova, Larissa B.; Sharpless, K. Barry; Powers, Evan T.; Wilson, Ian A.; Kelly, Jeffery W.Journal of the American Chemical Society (2013), 135 (15), 5656-5668CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Mols. that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) was conceived of by structure-based design principles and was chem. synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ∼1400 times faster than the hydrolysis reaction outside the thyroxine binding site. X-ray crystallog. confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. Eleven of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. Eleven of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore.
- 290Dalton, S. E.; Dittus, L.; Thomas, D. A.; Convery, M. A.; Nunes, J.; Bush, J. T.; Evans, J. P.; Werner, T.; Bantscheff, M.; Murphy, J. A.; Campos, S. Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition. J. Am. Chem. Soc. 2018, 140 (3), 932– 939, DOI: 10.1021/jacs.7b08979[ACS Full Text], [CAS], Google Scholar290https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFKitrrK&md5=90bb399303a942fb87f1b9096eb8bdc6Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase InhibitionDalton, Samuel E.; Dittus, Lars; Thomas, Daniel A.; Convery, Maire A.; Nunes, Joao; Bush, Jacob T.; Evans, John P.; Werner, Thilo; Bantscheff, Marcus; Murphy, John A.; Campos, SebastienJournal of the American Chemical Society (2018), 140 (3), 932-939CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to date in the development of chem. probes and approved drugs. However, this approach is limited to ∼200 kinases possessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing selective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compd. shows high levels of selectivity over a selection of lipid and protein kinases in biochem. assays, as well as covalent binding to very few off-target proteins in live-cell proteomic studies. We anticipate this approach could offer a general strategy, as an alternative to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.
- 291Gupta, R. C.; Sachana, M.; Mukherjee, I. M.; Doss, R. B.; Malik, J. K.; Milatovic, D. Organophosphates and Carbamates. In Veterinary Toxicology; Gupta, R. C., Ed.; Academic Press, 2018; pp. 495– 508, DOI: 10.1016/B978-0-12-811410-0.00037-4 .
- 292Tamura, T.; Ueda, T.; Goto, T.; Tsukidate, T.; Shapira, Y.; Nishikawa, Y.; Fujisawa, A.; Hamachi, I. Rapid Labelling and Covalent Inhibition of Intracellular Native Proteins Using Ligand-Directed N -Acyl- N -Alkyl Sulfonamide. Nat. Commun. 2018, 9 (1), 1870, DOI: 10.1038/s41467-018-04343-0[Crossref], [PubMed], [CAS], Google Scholar292https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MfjtFygug%253D%253D&md5=98b2280e760b0e170af05a6283c5d794Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamideTamura Tomonori; Ueda Tsuyoshi; Goto Taiki; Tsukidate Taku; Nishikawa Yuki; Fujisawa Alma; Hamachi Itaru; Shapira Yonatan; Hamachi ItaruNature communications (2018), 9 (1), 1870 ISSN:.Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~10(4) M(-1) s(-1), comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.
- 293Evans, M. J.; Saghatelian, A.; Sorensen, E. J.; Cravatt, B. F. Target Discovery in Small-Molecule Cell-Based Screens by in Situ Proteome Reactivity Profiling. Nat. Biotechnol. 2005, 23, 1303, DOI: 10.1038/nbt1149[Crossref], [PubMed], [CAS], Google Scholar293https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVOhu7nP&md5=1fc3d093fe8c7c7ccd7d610f10df105cTarget discovery in small-molecule cell-based screens by in situ proteome reactivity profilingEvans, Michael J.; Saghatelian, Alan; Sorensen, Erik J.; Cravatt, Benjamin F.Nature Biotechnology (2005), 23 (10), 1303-1307CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Chem. genomics aims to discover small mols. that affect biol. processes through the perturbation of protein function. However, detg. the protein targets of bioactive compds. remains a formidable challenge. The authors address this problem here through the creation of a natural product-inspired small-mol. library bearing protein-reactive elements. Cell-based screening identified a compd., MJE3 (I), that inhibits breast cancer cell proliferation. In situ proteome reactivity profiling revealed that MJE3, but not other library members, covalently labeled the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), resulting in enzyme inhibition. Interestingly, MJE3 labeling and inhibition of PGAM1 were obsd. exclusively in intact cells. These results support the hypothesis that cancer cells depend on glycolysis for viability and promote PGAM1 as a potential therapeutic target. More generally, the incorporation of protein-reactive compds. into chem. genomics screens offers a means to discover targets of bioactive small mols. in living systems, thereby enabling downstream mechanistic investigations.
- 294Evans, M. J.; Morris, G. M.; Wu, J.; Olson, A. J.; Sorensen, E. J.; Cravatt, B. F. Mechanistic and Structural Requirements for Active Site Labeling of Phosphoglycerate Mutase by Spiroepoxides. Mol. BioSyst. 2007, 3, 495– 506, DOI: 10.1039/b705113a[Crossref], [PubMed], [CAS], Google Scholar294https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXms1Wnt74%253D&md5=b69570bc329ddc6db39235b8c241d6c7Mechanistic and structural requirements for active site labeling of phosphoglycerate mutase by spiroepoxidesEvans, Michael J.; Morris, Garrett M.; Wu, Jane; Olson, Arthur J.; Sorensen, Erik J.; Cravatt, Benjamin F.Molecular BioSystems (2007), 3 (7), 495-506CODEN: MBOIBW; ISSN:1742-206X. (Royal Society of Chemistry)We recently reported the pharmacol. screening of a natural products-inspired library of spiroepoxide probes, resulting in the discovery of an agent MJE3 that displayed anti-proliferative effects in human breast cancer cells. MJE3 was found to covalently inactivate phosphoglycerate mutase-1 (PGAM1), a glycolytic enzyme with postulated roles in cancer cell metab. and proliferation. Considering that MJE3 is one of the first examples of a cell-permeable, small-mol. inhibitor for PGAM1, we pursued a detailed examn. of its mechanism and structural requirements for covalent inactivation. MJE3 was found to label PGAM1 on lysine-100, a conserved active site residue implicated in substrate recognition. Structural features of MJE3 important for PGAM1 labeling included two key recognition elements (an indole ring and carboxylic acid), the stereochem. orientation of the spiroepoxide, and presentation of these various binding/reactive groups on a rigid cyclohexane scaffold. Modeling studies of the docked MJE3-PGAM1 complex provide a structural rationale for these stringent requirements. Overall, these studies indicate that a special combination of binding and reactive elements are united in the MJE3 structure to inactivate PGAM1. More generally, our findings provide further evidence that useful pharmacol. tools can emerge from screening structurally diverse libraries of protein-reactive probes.
- 295Bongard, J.; Lorenz, M.; Vetter, I. R.; Stege, P.; Porfetye, A. T.; Schmitz, A. L.; Kaschani, F.; Wolf, A.; Koch, U.; Nussbaumer, P.; Klebl, B.; Kaiser, M.; Ehrmann, M. Identification of Noncatalytic Lysine Residues from Allosteric Circuits via Covalent Probes. ACS Chem. Biol. 2018, 13 (5), 1307– 1312, DOI: 10.1021/acschembio.8b00101[ACS Full Text], [CAS], Google Scholar295https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnsFGisLo%253D&md5=b791163873aa49010b449540aab5be32Identification of Noncatalytic Lysine Residues from Allosteric Circuits via Covalent ProbesBongard, Jens; Lorenz, Marian; Vetter, Ingrid R.; Stege, Patricia; Porfetye, Arthur T.; Schmitz, Anna Laura; Kaschani, Farnusch; Wolf, Alex; Koch, Uwe; Nussbaumer, Peter; Klebl, Bert; Kaiser, Markus; Ehrmann, MichaelACS Chemical Biology (2018), 13 (5), 1307-1312CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Covalent modifications of nonactive site lysine residues by small mol. probes has recently evolved into an important strategy for interrogating biol. systems. Here, we report the discovery of a class of bioreactive compds. that covalently modify lysine residues in DegS, the rate limiting protease of the essential bacterial outer membrane stress response pathway. These modifications lead to an allosteric activation and allow the identification of novel residues involved in the allosteric activation circuit. These findings were validated by structural analyses via X-ray crystallog. and cell-based reporter systems. We anticipate that our findings are not only relevant for a deeper understanding of the structural basis of allosteric activation in DegS and other HtrA serine proteases but also pinpoint an alternative use of covalent small mols. for probing essential biochem. mechanisms.
- 296Diethelm, S.; Schafroth, M. A.; Carreira, E. M. Amine-Selective Bioconjugation Using Arene Diazonium Salts. Org. Lett. 2014, 16 (15), 3908– 3911, DOI: 10.1021/ol5016509[ACS Full Text], [CAS], Google Scholar296https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFCrtbfF&md5=91876c83f67d31189c501b567630900eAmine-Selective Bioconjugation Using Arene Diazonium SaltsDiethelm, Stefan; Schafroth, Michael A.; Carreira, Erick M.Organic Letters (2014), 16 (15), 3908-3911CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)A novel bioconjugation strategy is presented that relies on the coupling of diazonium terephthalates with amines in proteins. The diazonium captures the amine while the vicinal ester locks it through cyclization, ensuring no reversibility. The reaction is highly efficient and proceeds under mild conditions and short reaction times. Densely functionalized, complex natural products were directly coupled to proteins using low concns. of coupling partners.
- 297Tung, C. L.; Wong, C. T. T.; Fung, E. Y. M.; Li, X. Traceless and Chemoselective Amine Bioconjugation via Phthalimidine Formation in Native Protein Modification. Org. Lett. 2016, 18 (11), 2600– 2603, DOI: 10.1021/acs.orglett.6b00983[ACS Full Text], [CAS], Google Scholar297https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XotFelsLs%253D&md5=93516d763bd6399a7753e7e5931d77e1Traceless and chemoselective amine bioconjugation via phthalimidine formation in native protein modificationTung, Chun Ling; Wong, Clarence T. T.; Fung, Eva Yi Man; Li, XuechenOrganic Letters (2016), 18 (11), 2600-2603CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)Ortho-Phthalaldehyde (OPA) and its derivs. are found to react chemoselectively with amino groups on peptides and proteins rapidly and tracelessly under the physiol. condition via formation of phthalimidines, which provides a novel and promising approach when performing bioconjugation on native proteins. The notable advantages of this method over the existing native protein lysine-labeling approaches include a traceless process, a self-reacting, specific and fast reaction, ease of operation, and the ability to use nonhydrolyzable reagents. Its applications have been effectively demonstrated including conjugation of peptides and proteins, and generation of an active PEGlyated L-asparaginase.
- 298Ritter, E.; Przybylski, P.; Brzezinski, B.; Bartl, F. Schiff Bases in Biological Systems. Curr. Org. Chem. 2009, 13 (3), 241– 249, DOI: 10.2174/138527209787314805[Crossref], [CAS], Google Scholar298https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXkslCnu7Y%253D&md5=d09e6ac15bea3a2959af966aef7bc7feSchiff bases in biological systemsRitter, Eglof; Przybylski, Piotr; Brzezinski, Bogumil; Bartl, FranzCurrent Organic Chemistry (2009), 13 (3), 241-249CODEN: CORCFE; ISSN:1385-2728. (Bentham Science Publishers Ltd.)A review. In many cases, the cofactors of biol. important proteins are linked to their apoproteins by Schiff bases. In this review, we discuss three classes of proteins in which Schiff bases are crucial for their function and catalytic mechanisms. First, we focus on retinylidene proteins, in which the cofactor retinal is fixed to the apoprotein by a protonated Schiff base. Light-induced retinal isomerization triggers a photocycle or a cascade of structural changes in which deprotonation of the Schiff base is a crucial step. We compare the photocycles of archaeal type rhodopsins, channelrhodopsins and the light induced cascades of visual rhodopsins. Pyridoxal phosphate (PLP) dependent enzymes are summarized in the second section. In these proteins, the cofactor PLP is linked via a Schiff base to the side chain of a lysine. In most cases, hydrolysis of this linkage is crit. for substrate binding. Finally, aldolases, that catalyze aldol cleavage during glycolysis, contain a lysine residue at their active center. Formation of a protonated Schiff base linkage to a carbonyl group of the substrate is the first step of the catalytic mechanism. This will be discussed in the last section.
- 299Malátková, P.; Wsól, V. Carbonyl Reduction Pathways in Drug Metabolism. Drug Metab. Rev. 2014, 46 (1), 96– 123, DOI: 10.3109/03602532.2013.853078[Crossref], [PubMed], [CAS], Google Scholar299https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVyksr8%253D&md5=9e605b6554b02cfb016c191128ba9e67Carbonyl reduction pathways in drug metabolismMalatkova, Petra; Wsol, VladimirDrug Metabolism Reviews (2014), 46 (1), 96-123CODEN: DMTRAR; ISSN:0360-2532. (Informa Healthcare)A review. The understanding of drug biotransformation is an important medical topic. The oxidative pathways that involve CYPs have been extensively studied in drug metab. in contrast to the reductive pathways. This review focuses on drugs that have been reported to be reduced at the carbonyl group in vivo. Although the carbonyl redn. of these drugs is well known, our understanding of the carbonyl reducing enzymes (CRE) that perform these reactions is limited. We have summarized the published data in order to thoroughly describe the reductive metab. of the selected drugs and to demonstrate the role of carbonyl redn. in the context of their overall metab. The no. of drugs recognized as substrates for CREs has increased considerably in recent years. Moreover, the importance of carbonyl redn. in the overall metab. of these drugs is often surprisingly high. Because only limited information is available about the CREs responsible for these reactions, addnl. research is needed to improve our understanding of the metab. of drugs undergoing carbonyl redn. Carbonyl redn. should be investigated during drug development because it can either pos. or neg. influence drug efficacy.
- 300Cal, P. M. S. D.; Vicente, J. B.; Pires, E.; Coelho, A. V.; Veiros, L. F.; Cordeiro, C.; Gois, P. M. P. Iminoboronates: A New Strategy for Reversible Protein Modification. J. Am. Chem. Soc. 2012, 134 (24), 10299– 10305, DOI: 10.1021/ja303436y[ACS Full Text], [CAS], Google Scholar300https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnsF2gurY%253D&md5=ad8e9263660d93401e1ccad61cd312dbIminoboronates: A new strategy for reversible protein modificationCal, Pedro M. S. D.; Vicente, Joao B.; Pires, Elisabete; Coelho, Ana V.; Veiros, Luis F.; Cordeiro, Carlos; Gois, Pedro M. P.Journal of the American Chemical Society (2012), 134 (24), 10299-10305CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein modification has entered the limelight of chem. and biol. sciences, since, by appending small mols. into proteins surfaces, fundamental biol. and biophys. processes may be studied and even modulated in a physiol. context. Herein the authors present a new strategy to modify the lysine's ε-amino group and the protein's N-terminal, based on the formation of stable iminoboronates in aq. media. This functionality enables the stable and complete modification of these amine groups, which can be reversible upon the addn. of fructose, dopamine, or glutathione. A detailed DFT study is also presented to rationalize the obsd. stability toward hydrolysis of the iminoboronate constructs.
- 301Adams, J.; Kauffman, M. Development of the Proteasome Inhibitor VelcadeTM (Bortezomib). Cancer Invest. 2004, 22 (2), 304– 311, DOI: 10.1081/CNV-120030218[Crossref], [PubMed], [CAS], Google Scholar301https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjtl2qtb0%253D&md5=6448e87e5e90eecf7399342bdbf50979Development of the Proteasome Inhibitor Velcade (Bortezomib)Adams, Julian; Kauffman, MichaelCancer Investigation (2004), 22 (2), 304-311CODEN: CINVD7; ISSN:0735-7907. (Marcel Dekker, Inc.)A review. The dipeptide boronic acid analog VELCADE (Bortezomib; formerly known as PS-341, LDP-341 and MLM341) is a potent and selective inhibitor of the proteasome, a multicatalytic enzyme that mediates many cellular regulatory signals by degrading regulatory proteins or their inhibitors. The proteasome is, thus, a potential target for pharmacol. agents. Bortezomib, the first proteasome inhibitor to reach clin. trials, has shown in vitro and in vivo activity against a variety of malignancies, including myeloma, chronic lymphocytic leukemia, prostate cancer, pancreatic cancer, and colon cancer. The drug is rapidly cleared from the vascular compartment, but a novel pharmacodynamic assay has shown that bortezomib-mediated proteasome blockade is dose-dependent and reversible. Based on phase I studies demonstrating that bortezomib has manageable toxicities in patients with advanced cancers, phase II trials have been initiated for both solid and hematol. malignancies.
- 302Bandyopadhyay, A.; McCarthy, K. A.; Kelly, M. A.; Gao, J. Targeting Bacteria via Iminoboronate Chemistry of Amine-Presenting Lipids. Nat. Commun. 2015, 6, 6561, DOI: 10.1038/ncomms7561[Crossref], [PubMed], [CAS], Google Scholar302https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXotlahsrY%253D&md5=c5e382b0d72f27f7911694d743322da9Targeting bacteria via iminoboronate chemistry of amine-presenting lipidsBandyopadhyay, Anupam; McCarthy, Kelly A.; Kelly, Michael A.; Gao, JianminNature Communications (2015), 6 (), 6561CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Synthetic mols. that target specific lipids serve as powerful tools for understanding membrane biol. and may also enable new applications in biotechnol. and medicine. For example, selective recognition of bacterial lipids may give rise to novel antibiotics, as well as diagnostic methods for bacterial infection. Currently known lipid-binding mols. primarily rely on noncovalent interactions to achieve lipid selectivity. Here we show that targeted recognition of lipids can be realized by selectively modifying the lipid of interest via covalent bond formation. Specifically, we report an unnatural amino acid that preferentially labels amine-presenting lipids via iminoboronate formation under physiol. conditions. By targeting phosphatidylethanolamine and lysylphosphatidylglycerol, the two lipids enriched on bacterial cell surfaces, the iminoboronate chem. allows potent labeling of Gram-pos. bacteria even in the presence of 10% serum, while bypassing mammalian cells and Gram-neg. bacteria. The covalent strategy for lipid recognition should be extendable to other important membrane lipids.
- 303Bandyopadhyay, A.; Gao, J. Iminoboronate Formation Leads to Fast and Reversible Conjugation Chemistry of α-Nucleophiles at Neutral PH. Chem. - Eur. J. 2015, 21 (42), 14748– 14752, DOI: 10.1002/chem.201502077[Crossref], [PubMed], [CAS], Google Scholar303https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVSis7zF&md5=03b9d05efc18ebe7534549609ccc5361Iminoboronate Formation Leads to Fast and Reversible Conjugation Chemistry of α-Nucleophiles at Neutral pHBandyopadhyay, Anupam; Gao, JianminChemistry - A European Journal (2015), 21 (42), 14748-14752CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)Bioorthogonal reactions that are fast and reversible under physiol. conditions are in high demand for biol. applications. Herein, an ortho boronic acid substituent makes aryl ketones rapidly conjugate with α-nucleophiles at neutral pH. Specifically, 2-acetylphenylboronic acid and derivs. conjugate with phenylhydrazine with rate consts. of 102 to 103 M-1s-1, comparable to the fastest bioorthogonal conjugations known to date. 11B NMR anal. revealed the varied extent of iminoboronate formation of the conjugates, in which the imine nitrogen forms a dative bond with boron. The iminoboronate formation activates the imines for hydrolysis and exchange, rendering these oxime/hydrazone conjugations reversible and dynamic under physiol. conditions. The fast and dynamic nature of the iminoboronate chem. should find wide applications in biol.
- 304Akçay, G.; Belmonte, M. A.; Aquila, B.; Chuaqui, C.; Hird, A. W.; Lamb, M. L.; Rawlins, P. B.; Su, N.; Tentarelli, S.; Grimster, N. P.; Su, Q. Inhibition of Mcl-1 through Covalent Modification of a Noncatalytic Lysine Side Chain. Nat. Chem. Biol. 2016, 12 (11), 931– 936, DOI: 10.1038/nchembio.2174[Crossref], [PubMed], [CAS], Google Scholar304https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKqtb3P&md5=bda5720579a9b5e6027632d2cffba2e1Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chainAkcay, Gizem; Belmonte, Matthew A.; Aquila, Brian; Chuaqui, Claudio; Hird, Alexander W.; Lamb, Michelle L.; Rawlins, Philip B.; Su, Nancy; Tentarelli, Sharon; Grimster, Neil P.; Su, QibinNature Chemical Biology (2016), 12 (11), 931-936CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Targeted covalent inhibition of disease-assocd. proteins has become a powerful methodol. in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here the authors used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to the knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chem. strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochem. and cell-based assays. The authors identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biol. phenomena.
- 305Harris, T. K.; Turner, G. J. Structural Basis of Perturbed PKa Values of Catalytic Groups in Enzyme Active Sites. IUBMB Life 2002, 53 (2), 85– 98, DOI: 10.1080/15216540211468[Crossref], [PubMed], [CAS], Google Scholar305https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XktlGisL8%253D&md5=fd5c17ecf362bb5d16790fbea204d512Structural basis of perturbed pKa values of catalytic groups in enzyme active sitesHarris, Thomas K.; Turner, George J.IUBMB Life (2002), 53 (2), 85-98CODEN: IULIF8; ISSN:1521-6543. (Taylor & Francis Inc.)A review with 81 refs. In protein and RNA macromols., only a limited no. of different side-chain chem. groups are available to function as catalysts. The myriad of enzyme-catalyzed reactions results from the ability of most of these groups to function either as nucleophilic, electrophilic, or general acid-base catalysts, and the key to their adapted chem. function lies in their states of protonation. Ionization is detd. by the intrinsic pKa of the group and the microenvironment created around the group by the protein or RNA structure, which perturbs its intrinsic pKa to its functional or apparent pKa. These pKa shifts result from interactions of the catalytic group with other fully or partially charged groups as well as the polarity or dielec. of the medium that surrounds it. The electrostatic interactions between ionizable groups found on the surface of macromols. are weak and cause only slight pKa perturbations (<2 units). The sum of many of these weak electrostatic interactions helps contribute to the stability of native or folded macromols. and their ligand complexes. However, the pKa values of catalytic groups that are found in the active sites of numerous enzymes are significantly more perturbed (>2 units) and are the subject of this review. The magnitudes of these pKa perturbations are analyzed with respect to the structural details of the active site microenvironment and the energetics of the reactions that they catalyze.
- 306Schwans, J. P.; Sunden, F.; Gonzalez, A.; Tsai, Y.; Herschlag, D. Uncovering the Determinants of a Highly Perturbed Tyrosine PKa in the Active Site of Ketosteroid Isomerase. Biochemistry 2013, 52 (44), 7840– 7855, DOI: 10.1021/bi401083b[ACS Full Text], [CAS], Google Scholar306https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFehu7vK&md5=2b02ad3af9f63850628edd6355beb7e1Uncovering the Determinants of a Highly Perturbed Tyrosine pKa in the Active Site of Ketosteroid IsomeraseSchwans, Jason P.; Sunden, Fanny; Gonzalez, Ana; Tsai, Yingssu; Herschlag, DanielBiochemistry (2013), 52 (44), 7840-7855CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Within the idiosyncratic enzyme active-site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aq. soln. Whereas structural inspection of systems has often attributed perturbed pKa values to dominant contributions from placement near charged groups or within hydrophobic pockets, Tyr57 of a Pseudomonas putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semisynthesis, 13C NMR spectroscopy, absorbance spectroscopy, and X-ray crystallog., was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of pos. charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from the introduction of nearby anionic groups, arises from accompanying active-site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water mol. that is part of a stabilizing hydrogen-bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen-bond donors, two Tyr side chains, and a water mol. positioned by other side chains and by a water-mediated hydrogen-bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural anal. can greatly facilitate our understanding of enzyme active-site energetics. The extensive data set provided may also be a valuable resource for those wishing to extensively test computational approaches for detg. enzymic pKa values and energetic effects.
- 307Hett, E. C.; Xu, H.; Geoghegan, K. F.; Gopalsamy, A.; Kyne, R. E.; Menard, C. A.; Narayanan, A.; Parikh, M. D.; Liu, S.; Roberts, L.; Robinson, R. P.; Tones, M. A.; Jones, L. H. Rational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride Probes. ACS Chem. Biol. 2015, 10 (4), 1094– 1098, DOI: 10.1021/cb5009475[ACS Full Text], [CAS], Google Scholar307https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmt1Kqug%253D%253D&md5=4af0b5188be76f67d43f307287571fdfRational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride ProbesHett, Erik C.; Xu, Hua; Geoghegan, Kieran F.; Gopalsamy, Ariamala; Kyne, Robert E.; Menard, Carol A.; Narayanan, Arjun; Parikh, Mihir D.; Liu, Shenping; Roberts, Lee; Robinson, Ralph P.; Tones, Michael A.; Jones, Lyn H.ACS Chemical Biology (2015), 10 (4), 1094-1098CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)This work describes the first rational targeting of tyrosine residues in a protein binding site by small-mol. covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based mol. design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition expts. with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that were used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chem. biol. toolkit by these probes uses in target validation and mol. pharmacol.
- 308Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation. J. Am. Chem. Soc. 2004, 126 (49), 15942– 15943, DOI: 10.1021/ja0439017[ACS Full Text], [CAS], Google Scholar308https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXpvFWjur8%253D&md5=d9cb096caf0ad9046b42c8e6bdb7145aA Three-Component Mannich-Type Reaction for Selective Tyrosine BioconjugationJoshi, Neel S.; Whitaker, Leanna R.; Francis, Matthew B.Journal of the American Chemical Society (2004), 126 (49), 15942-15943CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A new selective bioconjugation reaction is described for the modification of tyrosine residues on protein substrates. The reaction uses imines formed in situ from aldehydes and electron-rich anilines to modify phenolic side chains through a Mannich-type electrophilic arom. substitution pathway. The reaction takes place under mild pH and temp. conditions and can modify protein substrates at concns. as low as 20 μM. Using an efficient fluorescence-based assay, we demonstrated the reaction using a no. of aldehydes and protein targets. Importantly, proteins lacking surface-accessible tyrosines remained unmodified. It was also demonstrated that enzymic activity is preserved under the mild reaction conditions. This strategy represents one of the first carbon-carbon bond-forming reactions for protein modification and provides an important complement to more commonly used lysine- and cysteine-based methods.
- 309Jones, M. W.; Mantovani, G.; Blindauer, C. A.; Ryan, S. M.; Wang, X.; Brayden, D. J.; Haddleton, D. M. Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium Salts. J. Am. Chem. Soc. 2012, 134 (17), 7406– 7413, DOI: 10.1021/ja211855q[ACS Full Text], [CAS], Google Scholar309https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xltl2gtLw%253D&md5=fb9846543b44d6852e9fb52ca0b8c72fDirect Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium SaltsJones, Mathew W.; Mantovani, Giuseppe; Blindauer, Claudia A.; Ryan, Sinead M.; Wang, Xuexuan; Brayden, David J.; Haddleton, David M.Journal of the American Chemical Society (2012), 134 (17), 7406-7413CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Direct polymer conjugation at peptide tyrosine residues is described. In this study Tyr residues of both leucine enkephalin and salmon calcitonin (sCT) were targeted using appropriate diazonium salt-terminated linear monomethoxy poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacrylate prepd. by atom transfer radical polymn. Judicious choice of the reaction conditions-pH, stoichiometry, and chem. structure of diazonium salt-led to a high degree of site-specificity in the conjugation reaction, even in the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed that conjugation of mPEG2000 to sCT did not affect the peptide's ability to increase intracellular cAMP induced in T47D human breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca2+] plasma levels by mPEG2000-sCT conjugate in rat animal models.
- 310Ban, H.; Gavrilyuk, J.; Barbas, C. F. Tyrosine Bioconjugation through Aqueous Ene-Type Reactions: A Click-Like Reaction for Tyrosine. J. Am. Chem. Soc. 2010, 132 (5), 1523– 1525, DOI: 10.1021/ja909062q[ACS Full Text], [CAS], Google Scholar310https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkt1Wrug%253D%253D&md5=876ee34e149ceed4f70f7fd4efbb25baTyrosine bioconjugation through aqueous ene-type reactions: A click-like reaction for tyrosineBan, Hitoshi; Gavrilyuk, Julia; Barbas, Carlos F.Journal of the American Chemical Society (2010), 132 (5), 1523-1525CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A new and versatile class of cyclic diazodicarboxamides that reacts efficiently and selectively with phenols and the phenolic side chain of tyrosine through an ene-like reaction is reported. This mild aq. tyrosine ligation reaction works over a broad pH range and expands the repertoire of aq. chemistries available for small mol., peptide, and protein modification. The tyrosine ligation reactions are shown to be compatible with the labeling of native enzymes and antibodies in a buffered aq. soln. This reaction provides a novel synthetic approach to bispecific antibodies. The authors believe this reaction will find broad utility in peptide and protein chem. and in the chem. of phenol-contg. compds.
- 311Ban, H.; Nagano, M.; Gavrilyuk, J.; Hakamata, W.; Inokuma, T.; Barbas, C. F. Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction. Bioconjugate Chem. 2013, 24 (4), 520– 532, DOI: 10.1021/bc300665t[ACS Full Text], [CAS], Google Scholar311https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXkslWlsrY%253D&md5=7f548b32c1e16dd72adbe15b05bbf38cFacile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click ReactionBan, Hitoshi; Nagano, Masanobu; Gavrilyuk, Julia; Hakamata, Wataru; Inokuma, Tsubasa; Barbas, Carlos F., IIIBioconjugate Chemistry (2013), 24 (4), 520-532CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivs. in peptide and protein chem., we synthesized PTAD derivs. possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivs. and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective trifunctionalization of proteins is readily achieved. In particular cases, we noted that PTAD decompn. resulted in formation of a putative isocyanate byproduct that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addn. of a small amt. of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene glycol) chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen, whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody-drug conjugate. For this purpose, we synthesized a PTAD deriv. linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small mols. The tyrosine click linkage demonstrated stability to extremes of pH, temp., and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small mols., peptides, and proteins under mild aq. conditions over a broad pH range using a wide variety of biol. acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compns.
- 312Hatcher, J. M.; Wu, G.; Zeng, C.; Zhu, J.; Meng, F.; Patel, S.; Wang, W.; Ficarro, S. B.; Leggett, A. L.; Powell, C. E.; Marto, J. A.; Zhang, K.; Ngo, J. C. K.; Fu, X.-D.; Zhang, T.; Gray, N. S. SRPKIN-1: A Covalent SRPK1/2 Inhibitor That Potently Converts VEGF from Pro-Angiogenic to Anti-Angiogenic Isoform. Cell Chem. Biol. 2018, 25, 460– 470, DOI: 10.1016/j.chembiol.2018.01.013[Crossref], [PubMed], [CAS], Google Scholar312https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjt1Sgtrs%253D&md5=713e21a6f41894c0b0a0c876ef5ded79SRPKIN-1: A Covalent SRPK1/2 Inhibitor that Potently Converts VEGF from Pro-angiogenic to Anti-angiogenic IsoformHatcher, John M.; Wu, Guowei; Zeng, Chuyue; Zhu, Jie; Meng, Fan; Patel, Sherrina; Wang, Wenqiu; Ficarro, Scott B.; Leggett, Alan L.; Powell, Chelsea E.; Marto, Jarrod A.; Zhang, Kang; Ki Ngo, Jacky Chi; Fu, Xiang-Dong; Zhang, Tinghu; Gray, Nathanael S.Cell Chemical Biology (2018), 25 (4), 460-470.e6CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)The SRPK family of kinases regulates pre-mRNA splicing by phosphorylating serine/arginine (SR)-rich splicing factors, signals splicing control in response to extracellular stimuli, and contributes to tumorigenesis, suggesting that these splicing kinases are potential therapeutic targets. Here, we report the development of the first irreversible SRPK inhibitor, SRPKIN-1, which is also the first kinase inhibitor that forms a covalent bond with a tyrosine phenol group in the ATP-binding pocket. Kinome-wide profiling demonstrates its selectivity for SRPK1/2, and SRPKIN-1 attenuates SR protein phosphorylation at submicromolar concns. Vascular endothelial growth factor (VEGF) is a known target for SRPK-regulated splicing and, relative to the first-generation SRPK inhibitor SRPIN340 or small interfering RNA-mediated SRPK knockdown, SRPKIN-1 is more potent in converting the pro-angiogenic VEGF-A165a to the anti-angiogenic VEGF-A165b isoform and in blocking laser-induced neovascularization in a murine retinal model. These findings encourage further development of SRPK inhibitors for treatment of age-related macular degeneration.
- 313Patricelli, M. P.; Nomanbhoy, T. K.; Wu, J.; Brown, H.; Zhou, D.; Zhang, J.; Jagannathan, S.; Aban, A.; Okerberg, E.; Herring, C.; Nordin, B.; Weissig, H.; Yang, Q.; Lee, J.-D.; Gray, N. S.; Kozarich, J. W. In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases. Chem. Biol. 2011, 18 (6), 699– 710, DOI: 10.1016/j.chembiol.2011.04.011[Crossref], [PubMed], [CAS], Google Scholar313https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXotVejtbo%253D&md5=dda091b895850c95131f80c3aba9e8c7In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native KinasesPatricelli, Matthew P.; Nomanbhoy, Tyzoon K.; Wu, Jiangyue; Brown, Heidi; Zhou, David; Zhang, Jianming; Jagannathan, Subadhra; Aban, Arwin; Okerberg, Eric; Herring, Chris; Nordin, Brian; Weissig, Helge; Yang, Qingkai; Lee, Jiing-Dwan; Gray, Nathanael S.; Kozarich, John W.Chemistry & Biology (Cambridge, MA, United States) (2011), 18 (6), 699-710CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Summary: Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biol. targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Addnl., Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.
- 314Sakamoto, H.; Tsukaguchi, T.; Hiroshima, S.; Kodama, T.; Kobayashi, T.; Fukami, T. A.; Oikawa, N.; Tsukuda, T.; Ishii, N.; Aoki, Y. CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. Cancer Cell 2011, 19 (5), 679– 690, DOI: 10.1016/j.ccr.2011.04.004[Crossref], [PubMed], [CAS], Google Scholar314https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtFGlsL8%253D&md5=cae32f3a3647926ba835293b68e776f8CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper MutantSakamoto, Hiroshi; Tsukaguchi, Toshiyuki; Hiroshima, Sayuri; Kodama, Tatsushi; Kobayashi, Takamitsu; Fukami, Takaaki A.; Oikawa, Nobuhiro; Tsukuda, Takuo; Ishii, Nobuya; Aoki, YukoCancer Cell (2011), 19 (5), 679-690CODEN: CCAECI; ISSN:1535-6108. (Cell Press)Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers, following gene alterations such as chromosomal translocation, amplification, or point mutation. Here, the authors identified CH5424802, a potent, selective, and orally available ALK inhibitor with a unique chem. scaffold, showing preferential antitumor activity against cancers with gene alterations of ALK, such as non-small cell lung cancer (NSCLC) cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion in vitro and in vivo. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven cell growth. The authors' results support the potential for clin. evaluation of CH5424802 for the treatment of patients with ALK-driven tumors.
- 315Choi, E. J.; Jung, D.; Kim, J.-S.; Lee, Y.; Kim, B. M. Chemoselective Tyrosine Bioconjugation through Sulfate Click Reaction. Chem. - Eur. J. 2018, 24 (43), 10948– 10952, DOI: 10.1002/chem.201802380[Crossref], [PubMed], [CAS], Google Scholar315https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlCmt7vE&md5=f0bcca396c1439a2501cc04c98c9dde2Chemoselective Tyrosine Bioconjugation through Sulfate Click ReactionChoi, Eun Joung; Jung, Dongwook; Kim, Jong-Seo; Lee, Yan; Kim, B. MoonChemistry - A European Journal (2018), 24 (43), 10948-10952CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)A novel and selective tyrosine functionalization strategy through SuFEx (sulfur fluoride exchange) chem. is presented. In this approach, free tyrosine (Tyr) reacts selectively with aryl fluorosulfate in the presence of various nucleophilic amino acid residues in bio-tolerable conditions. Chemoselectivity of this unique SuFEx reaction was confirmed in amino acid, peptide, and protein conjugations. The functions of peptides and proteins were well-preserved as demonstrated from the Tyr-specific modification of cell-penetrating peptide and erythropoietin. This method is well-suited for residue-specific modification of native proteins, and thus would expand the versatility of bio-conjugation in protein chem.
- 316Fadeyi, O. O.; Hoth, L. R.; Choi, C.; Feng, X.; Gopalsamy, A.; Hett, E. C.; Kyne, R. E.; Robinson, R. P.; Jones, L. H. Covalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine Residue. ACS Chem. Biol. 2017, 12 (8), 2015– 2020, DOI: 10.1021/acschembio.7b00403[ACS Full Text], [CAS], Google Scholar316https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFyisLfI&md5=d9b49c1f7d95cba028a1eb7a9f8e776aCovalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine ResidueFadeyi, Olugbeminiyi O.; Hoth, Lise R.; Choi, Chulho; Feng, Xidong; Gopalsamy, Ariamala; Hett, Erik C.; Kyne, Robert E., Jr.; Robinson, Ralph P.; Jones, Lyn H.ACS Chemical Biology (2017), 12 (8), 2015-2020CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Irreversible enzyme inhibitors and covalent chem. biol. probes often utilize the reaction of a protein cysteine residue with an appropriately positioned electrophile (e.g., acrylamide) on the ligand template. However, cysteine residues are not always available for site-specific protein labeling, and therefore new approaches are needed to expand the toolkit of appropriate electrophiles ("warheads") that target alternative amino acids. We previously described the rational targeting of tyrosine residues in the active site of a protein (the mRNA decapping scavenger enzyme, DcpS) using inhibitors armed with a sulfonyl fluoride electrophile. These inhibitors subsequently enabled the development of clickable probe technol. to measure drug-target occupancy in live cells. Here we describe a fluorosulfate-contg. inhibitor (aryl fluorosulfate probe (FS-p1)) with excellent chem. and metabolic stability that reacts selectively with a noncatalytic serine residue in the same active site of DcpS as confirmed by peptide mapping expts. Our results suggest that noncatalytic serine targeting using fluorosulfate electrophilic warheads could be a suitable strategy for the development of covalent inhibitor drugs and chem. probes.
- 317Crawford, L. A.; Weerapana, E. A Tyrosine-Reactive Irreversible Inhibitor for Glutathione S-Transferase Pi (GSTP1). Mol. BioSyst. 2016, 12 (6), 1768– 1771, DOI: 10.1039/C6MB00250A[Crossref], [PubMed], [CAS], Google Scholar317https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsF2ntb8%253D&md5=f30c8260bb5e863fe8f0d70fb3aef15dA tyrosine-reactive irreversible inhibitor for glutathione S-transferase Pi (GSTP1)Crawford, L. A.; Weerapana, E.Molecular BioSystems (2016), 12 (6), 1768-1771CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)Glutathione S-transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, the authors report LAS17, a dichlorotriazine-contg. compd. that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition.
- 318Gehringer, M.; Forster, M.; Pfaffenrot, E.; Bauer, S. M.; Laufer, S. A. Novel Hinge-Binding Motifs for Janus Kinase 3 Inhibitors: A Comprehensive Structure–Activity Relationship Study on Tofacitinib Bioisosteres. ChemMedChem 2014, 9 (11), 2516– 2527, DOI: 10.1002/cmdc.201402252[Crossref], [PubMed], [CAS], Google Scholar318https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlOksrnE&md5=f1ca6c4bf272b41b5204b8b82ea5836eNovel hinge-binding motifs for janus kinase 3 inhibitors: a comprehensive structure-activity relationship study on tofacitinib bioisosteresGehringer, Matthias; Forster, Michael; Pfaffenrot, Ellen; Bauer, Silke M.; Laufer, Stefan A.ChemMedChem (2014), 9 (11), 2516-2527CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The Janus kinases (JAKs) are a family of cytosolic tyrosine kinases crucially involved in cytokine signaling. JAKs have been demonstrated to be valid targets in the treatment of inflammatory and myeloproliferative disorders, and two inhibitors, tofacitinib and ruxolitinib, recently received their marketing authorization. Despite this success, selectivity within the JAK family remains a major issue. Both approved compds. share a common 7H-pyrrolo[2,3-d]pyrimidine hinge binding motif, and little is known about modifications tolerated at this heterocyclic core. In the current study, a library of tofacitinib bioisosteres was prepd. and tested against JAK3. The compds. possessed the tofacitinib piperidinyl side chain, whereas the hinge binding motif was replaced by a variety of heterocycles mimicking its pharmacophore. In view of the promising expectations obtained from mol. modeling, most of the compds. proved to be poorly active. However, strategies for restoring activity within this series of novel chemotypes were discovered and crucial structure-activity relationships were deduced. The compds. presented may serve as starting point for developing novel JAK inhibitors and as a valuable training set for in silico models.
- 319Gu, C.; Shannon, D. A.; Colby, T.; Wang, Z.; Shabab, M.; Kumari, S.; Villamor, J. G.; McLaughlin, C. J.; Weerapana, E.; Kaiser, M.; Cravatt, B. F.; van der Hoorn, R. A. L. Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases. Chem. Biol. 2013, 20 (4), 541– 548, DOI: 10.1016/j.chembiol.2013.01.016[Crossref], [PubMed], [CAS], Google Scholar319https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXmt1Clsbo%253D&md5=71d3ce60eed76d8a361fc0057d2102e1Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione TransferasesGu, Christian; Shannon, D. Alexander; Colby, Tom; Wang, Zheming; Shabab, Mohammed; Kumari, Selva; Villamor, Joji Grace; McLaughlin, Christopher J.; Weerapana, Eranthie; Kaiser, Markus; Cravatt, Benjamin F.; van der Hoorn, Renier A. L.Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (4), 541-548CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)Chem. probes have great potential for identifying functional residues in proteins in crude proteomes. Here we studied labeling sites of chem. probes based on sulfonyl fluorides (SFs) on plant and animal proteomes. Besides serine proteases and many other proteins, SF-based probes label Tyr residues in glutathione transferases (GSTs). The labeled GSTs represent four different GST classes that share less than 30% sequence identity. The targeted Tyr residues are located at similar positions in the promiscuous substrate binding site and are essential for GST function. The high selectivity of SF-based probes for functional Tyr residues in GSTs illustrates how these probes can be used for functional studies of GSTs and other proteins in crude proteomes.
- 320Ekici, Ö. D.; Paetzel, M.; Dalbey, R. E. Unconventional Serine Proteases: Variations on the Catalytic Ser/His/Asp Triad Configuration. Protein Sci. 2008, 17 (12), 2023– 2037, DOI: 10.1110/ps.035436.108[Crossref], [PubMed], [CAS], Google Scholar320https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVKgt7rP&md5=ad4e54f8dd98af7f51999678806d40acUnconventional serine proteases: variations on the catalytic Ser/His/Asp triad configurationEkici, Ozlem Dogan; Paetzel, Mark; Dalbey, Ross E.Protein Science (2008), 17 (12), 2023-2037CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)A review. Serine proteases comprise nearly 1/3 of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called "classical" catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of "nonclassical" serine proteases where variations to the catalytic triad are obsd. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine proteases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. Here, the authors discuss the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.
- 321Long, J. Z.; Cravatt, B. F. The Metabolic Serine Hydrolases and Their Functions in Mammalian Physiology and Disease. Chem. Rev. 2011, 111 (10), 6022– 6063, DOI: 10.1021/cr200075y[ACS Full Text], [CAS], Google Scholar321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvFagtb8%253D&md5=c978f77bd911d5877df4ba97fc09e4b3The metabolic serine hydrolases and their functions in mammalian physiology and diseaseLong, Jonathan Z.; Cravatt, Benjamin F.Chemical Reviews (Washington, DC, United States) (2011), 111 (10), 6022-6063CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Serine hydrolases (SHs) consist of >200 enzymes in humans which are characterized by the presence of an active site Ser residue that is used for the hydrolysis of substrates. The membership of this enzyme class is nearly equally split between serine proteases (trypsin/chymotrypsin/subtilisin enzymes) and "metabolic" SHs that cleave ester, amide, or thioester bonds in small mols., peptides, or proteins. The focus of this review is on the metabolic SHs. Here, the authors attempt to provide a comprehensive summary that captures the state of knowledge about metabolic SHs in their entirety, including those enzymes that remain mostly or completely uncharacterized. Particular emphasis is placed on relating the biochem. and enzymol. of individual SHs to the physiol. substrates and products they regulate in living systems, and how SHs, through the regulation of specific metabolic pathways impact health and disease.
- 322Lei, J.; Zhou, Y.; Xie, D.; Zhang, Y. Mechanistic Insights into a Classic Wonder Drug—Aspirin. J. Am. Chem. Soc. 2015, 137 (1), 70– 73, DOI: 10.1021/ja5112964[ACS Full Text], [CAS], Google Scholar322https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFOgsLzI&md5=ec731b98115bd7b896b23d97087b24c3Mechanistic Insights into a Classic Wonder Drug-AspirinLei, Jinping; Zhou, Yanzi; Xie, Daiqian; Zhang, YingkaiJournal of the American Chemical Society (2015), 137 (1), 70-73CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Aspirin, one of the oldest and most common anti-inflammatory agents, has recently been shown to reduce cancer risks. The principal pharmacol. effects of aspirin are known to arise from its covalent modification of cyclooxygenase-2 (COX-2) through acetylation of Ser530, but the detailed mechanism of its biochem. action and specificity remains to be elucidated. In this work, we have filled this gap by employing a state-of-the-art computational approach, Born-Oppenheimer mol. dynamics simulations with ab initio quantum mech./mol. mech. potential and umbrella sampling. Our studies have characterized a substrate-assisted inhibition mechanism for aspirin acetylating COX: it proceeds in two successive stages with a metastable tetrahedral intermediate, in which the carboxyl group of aspirin serves as the general base. The computational results confirmed that aspirin would be 10-100 times more potent against COX-1 than against COX-2, and revealed that this inhibition specificity between the two COX isoforms can be attributed mainly to the difference in kinetics rate of the covalent inhibition reaction, not the aspirin-binding step. The structural origin of this differential inhibition of the COX enzymes by aspirin has also been elucidated.
- 323Leney, A. C.; Heck, A. J. R. Native Mass Spectrometry: What Is in the Name?. J. Am. Soc. Mass Spectrom. 2017, 28 (1), 5– 13, DOI: 10.1007/s13361-016-1545-3[ACS Full Text], [CAS], Google Scholar323https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFylsLzF&md5=09d56e0a888ad31a6f655c4e1d840775Native Mass Spectrometry: What is in the Name?Leney, Aneika C.; Heck, Albert J. R.Journal of the American Society for Mass Spectrometry (2017), 28 (1), 5-13CODEN: JAMSEF; ISSN:1044-0305. (Springer)A review. Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomol. mass spectrometry and proteomics. Advances in sample prepn. and mass analyzers have enabled researchers to ext. much more information from biol. samples than just the mol. wt. In particular, relevant for structural biol., noncovalent protein-protein and protein-ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomol. mass spectrometry, nowadays often referred to as "native MS," has come to maturation over the last two decades, with dozens of labs. using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure-function relations. In this perspective, the authors describe the origins of and (re)define the term native MS, portraying in detail what the authors meant by "native MS," when the term was coined and also describing what it does (according to the authors) not entail. Addnl., the authors describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technol. has in solving complex biol. questions.
- 324Li, Z.; Qian, L.; Li, L.; Bernhammer, J. C.; Huynh, H. V.; Lee, J.-S.; Yao, S. Q. Tetrazole Photoclick Chemistry: Reinvestigating Its Suitability as a Bioorthogonal Reaction and Potential Applications. Angew. Chem., Int. Ed. 2016, 55 (6), 2002– 2006, DOI: 10.1002/anie.201508104[Crossref], [CAS], Google Scholar324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFKisbjM&md5=6438d371472471f5e43159dc60712774Tetrazole Photoclick Chemistry: Reinvestigating Its Suitability as a Bioorthogonal Reaction and Potential ApplicationsLi, Zhengqiu; Qian, Linghui; Li, Lin; Bernhammer, Jan C.; Huynh, Han Vinh; Lee, Jun-Seok; Yao, Shao Q.Angewandte Chemie, International Edition (2016), 55 (6), 2002-2006CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The bioorthogonality of tetrazole photoclick chem. has been reassessed. Upon photolysis of a tetrazole, the highly reactive nitrile imine formed undergoes rapid nucleophilic reaction with a variety of nucleophiles present in a biol. system, along with the expected cycloaddn. with alkenes. The alternative use of the tetrazole photoclick reaction was thus explored: tetrazoles were incorporated into Bodipy and Acedan dyes, providing novel photo-crosslinkers with one- and two-photon fluorescence Turn-ON properties that may be developed into protein-detecting biosensors. Further introduction of these photo-activatable, fluorogenic moieties into staurosporine resulted in the corresponding probes capable of photoinduced, no-wash imaging of endogenous kinase activities in live mammalian cells.
- 325Zhao, S.; Dai, J.; Hu, M.; Liu, C.; Meng, R.; Liu, X.; Wang, C.; Luo, T. Photo-Induced Coupling Reactions of Tetrazoles with Carboxylic Acids in Aqueous Solution: Application in Protein Labelling. Chem. Commun. 2016, 52 (25), 4702– 4705, DOI: 10.1039/C5CC10445A[Crossref], [PubMed], [CAS], Google Scholar325https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs1ejs78%253D&md5=09c69da8265c035f3dcd8000d298c0b8Photo-induced coupling reactions of tetrazoles with carboxylic acids in aqueous solution: application in protein labelingZhao, Shan; Dai, Jianye; Hu, Mo; Liu, Chang; Meng, Rong; Liu, Xiaoyun; Wang, Chu; Luo, TuopingChemical Communications (Cambridge, United Kingdom) (2016), 52 (25), 4702-4705CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)The photo-induced reactions of diaryltetrazoles with carboxylic acids in aq. soln. were studied. Besides measuring the apparent second-order rate const. and evaluating the functional group compatibility of these reactions, the authors further incorporated the tetrazoles into SAHA, leading to a new active-site-directed probe for labeling HDACs in both cell lysates and living cells.
- 326Mix, K. A.; Raines, R. T. Optimized Diazo Scaffold for Protein Esterification. Org. Lett. 2015, 17 (10), 2358– 2361, DOI: 10.1021/acs.orglett.5b00840[ACS Full Text], [CAS], Google Scholar326https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXnvV2ktrc%253D&md5=5737bf6bf6f0a391613597bd2e4d906bOptimized Diazo Scaffold for Protein EsterificationMix, Kalie A.; Raines, Ronald T.Organic Letters (2015), 17 (10), 2358-2361CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)The O-alkylation of carboxylic acids with diazo compds. provides a means to esterify carboxylic acids in aq. soln. A Hammett anal. of the reactivity of diazo compds. derived from phenylglycinamide revealed that the (p-methylphenyl)glycinamide scaffold has an esp. high reaction rate and ester/alc. product ratio and esterifies protein carboxyl groups more efficiently than any known reagent.
- 327Ban, H. S.; Usui, T.; Nabeyama, W.; Morita, H.; Fukuzawa, K.; Nakamura, H. Discovery of Boron-Conjugated 4-Anilinoquinazoline as a Prolonged Inhibitor of EGFR Tyrosine Kinase. Org. Biomol. Chem. 2009, 7 (21), 4415– 4427, DOI: 10.1039/b909504g[Crossref], [PubMed], [CAS], Google Scholar327https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1Gksb%252FN&md5=b61ee61865bdff8e4706485b1f030554Discovery of boron-conjugated 4-anilinoquinazoline as a prolonged inhibitor of EGFR tyrosine kinaseBan, Hyun Seung; Usui, Taikou; Nabeyama, Wataru; Morita, Hidetoshi; Fukuzawa, Kaori; Nakamura, HiroyukiOrganic & Biomolecular Chemistry (2009), 7 (21), 4415-4427CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Boron-conjugated 4-anilinoquinazolines were designed and synthesized as inhibitors of EGFR tyrosine kinase with possible covalent bond interactions between the boron atom and the nucleophilic groups of the EGFR kinase domain. Among the compds. synthesized, compds. I and II (X = CH:CH, 1,4-phenylene) reduced the EGF-mediated phosphorylation of EGFR tyrosine kinase and its downstream kinases including ERK and Akt in A431 cells. The cell growth was inhibited by these compds. through arrest of G1 cell cycle, which induced apoptosis. A time-dependent in vitro preincubation assay demonstrated the irreversible inhibition of compd. II [X = 1,4-phenylene; (III)] against EGFR tyrosine kinase. Quantum mech. docking simulation revealed that the boronic acid moiety of compd. III formed a covalent B-O bond with Asp800 in addn. to hydrogen bonds with Asp800 and Cys797, which may cause the prolonged inhibition of compd. III toward EGFR tyrosine kinase.
- 328Woodward, R. B.; Olofson, R. A.; Mayer, H. A New Synthesis of Peptides. J. Am. Chem. Soc. 1961, 83 (4), 1010– 1012, DOI: 10.1021/ja01465a072[ACS Full Text], [CAS], Google Scholar328https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3MXnsFyhsQ%253D%253D&md5=52b956257b3ee4dff7701172de0a9d4cA new synthesis of peptidesWoodward, R. B.; Olofson, R. A.; Mayer, HansJournal of the American Chemical Society (1961), 83 (), 1010-12CODEN: JACSAT; ISSN:0002-7863.N-Ethyl-5-phenylisoxazolium-3'-sulfonate (I) reacted readily with carboxylates to serve as the carboxyl-activating step in a new simple synthesis of peptides. A protected amino acid or peptide (1 mole) dissolved in MeCN or MeNO2 contg. 1 mole Et3N was added to 1 mole I in the same solvent and stirred at 0° until soln. was complete (activation step). The amino acid ester-HCl or peptide ester-HCl (1 mole) and an equiv. amt. Et3N were added and stirred overnight at room temp. The solvent was removed and the mixt. worked up. Several examples were carried out with activation in MeCN at 0° for approx. 1 hr. (peptide, yield, m.p. given): L-PhCH2CH(NHZ)CONHCH2CO2Et, 93, 109-10°; o-C6H4(CO)2NCH2CONHCH2CO2Et, 88, 193.5-4.5°; L-ZHN(CH2)4CH(NHZ)CHCONHCH2CO2Et, 95, 90.5-2.5°; L-PhCH2CH(NHZ)CONHCH(CH2CHMe2)CO2Me-L, 90, 106-7°; L-ZHNCH(CH2CONH2)CONHCH2CO2Et (II), 79, 186-7°; L-PhCH2CH(HNOCCH2NHZ)CONHCH2CO2Et, 90, 116.5-7.5°; L-(4-HOC6H4)CH2CH(HNOCCH2NHOCCH2NHZ)CO2Me, 84, 159.5-61.5°; ZHNCH2CO(NHCH2CO)2OEt, 91, 167-8°; L-ZHNCH(CH2CH2SMe)CO(HNCH2CO)2OEt, 86, 131.5-3°; L-ZN.CH2.CH2.CH(OH).CHCO(HNCH2CO)2OEt, 80, 145-6°; and with activation in MeNO2 at room temp. for 7 min. (same data given): II, 80, 185.5-7.0°; L-ZHNCH(CH2CONH2)CONHCH(CH2CHMe2)CO2Me-L, 76, 176.5-8.0°; L-ZHNCH(CH2CH2CONH2)CONHCH(CHMe2)CO2Me-L, 77, 172.5-3.0°; L-ZHNCH(CH2CH2CONH2)CONHCH(CH2C6H4OH - 4)CO2Me-L, 75, 198-9°; ZHNCH2CO(HNCH2CO)2OEt, 90, 167-8°; DL-PhCH2CH(HNOCCH2NHZ)CONHCH2CO2Et, 89, 132-3°.
- 329Martín-Gago, P.; Fansa, E. K.; Winzker, M.; Murarka, S.; Janning, P.; Schultz-Fademrecht, C.; Baumann, M.; Wittinghofer, A.; Waldmann, H. Covalent Protein Labeling at Glutamic Acids. Cell Chem. Biol. 2017, 24, 589– 597, DOI: 10.1016/j.chembiol.2017.03.015[Crossref], [PubMed], [CAS], Google Scholar329https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmsVWiur4%253D&md5=82057dbd875a929697a36ff86c532160Covalent Protein Labeling at Glutamic AcidsMartin-Gago, Pablo; Fansa, Eyad K.; Winzker, Michael; Murarka, Sandip; Janning, Petra; Schultz-Fademrecht, Carsten; Baumann, Matthias; Wittinghofer, Alfred; Waldmann, HerbertCell Chemical Biology (2017), 24 (5), 589-597.e5CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Covalent labeling of amino acids in proteins by reactive small mols., in particular at cysteine SH and lysine NH groups, is a powerful approach to identify and characterize proteins and their functions. However, for the less-reactive carboxylic acids present in Asp and Glu, hardly any methodol. is available. Employing the lipoprotein binding chaperone PDE6δ as an example, we demonstrate that incorporation of isoxazolium salts that resemble the structure and reactivity of Woodward's reagent K into protein ligands provides a novel method for selective covalent targeting of binding site carboxylic acids in whole proteomes. Covalent adduct formation occurs via rapid formation of enol esters and the covalent bond is stable even in the presence of strong nucleophiles. This new method promises to open up hitherto unexplored opportunities for chem. biol. research.
- 330Martín-Gago, P.; Fansa, E. K.; Klein, C. H.; Murarka, S.; Janning, P.; Schürmann, M.; Metz, M.; Ismail, S.; Schultz-Fademrecht, C.; Baumann, M.; Bastiaens, P. I. H.; Wittinghofer, A.; Waldmann, H. A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity That Prevents Efficient Inhibitor Release by Arl2. Angew. Chem., Int. Ed. 2017, 56 (9), 2423– 2428, DOI: 10.1002/anie.201610957[Crossref], [CAS], Google Scholar330https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsValt7k%253D&md5=2b66846ac0d9806f58b428d83819a7c7A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2Martin-Gago, Pablo; Fansa, Eyad K.; Klein, Christian H.; Murarka, Sandip; Janning, Petra; Schuermann, Marc; Metz, Malte; Ismail, Shehab; Schultz-Fademrecht, Carsten; Baumann, Matthias; Bastiaens, Philippe I. H.; Wittinghofer, Alfred; Waldmann, HerbertAngewandte Chemie, International Edition (2017), 56 (9), 2423-2428CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Small-mol. inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro (KD<10 nM), interference with Ras signaling and growth inhibition require 5-20 μm compd. concns. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compds. selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas.
- 331Tsukiji, S.; Hamachi, I. Ligand-Directed Tosyl Chemistry for in Situ Native Protein Labeling and Engineering in Living Systems: From Basic Properties to Applications. Curr. Opin. Chem. Biol. 2014, 21, 136– 143, DOI: 10.1016/j.cbpa.2014.07.012[Crossref], [PubMed], [CAS], Google Scholar331https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVOhs7nK&md5=0e1580b982702ef2074672524209f5e2Ligand-directed tosyl chemistry for in situ native protein labeling and engineering in living systems: from basic properties to applicationsTsukiji, Shinya; Hamachi, ItaruCurrent Opinion in Chemical Biology (2014), 21 (), 136-143CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. The ability to introduce any chem. probe to any endogenous target protein in its native environment, that is in cells and in vivo, is anticipated to provide various new exciting tools for biol. and biomedical research. Although still at the prototype stage, the ligand-directed tosyl (LDT) chem. is a novel type of affinity labeling technique that we developed for such a dream. This chem. allows for modifying native proteins by various chem. probes with high specificity in various biol. settings ranging from in vitro (in test tubes) to in living cells and in vivo. Since the first report, the list of proteins that are successfully labeled by the LDT chem. has been increasing. A growing no. of studies have demonstrated its utility to create semisynthetic proteins directly in cellular contexts. The in situ generated semisynthetic proteins are applicable for various types of anal. and imaging of intracellular biol. processes. In this review, we summarize the basic properties of the LDT chem. and its applications toward in situ engineering and anal. of native proteins in living systems. Current limitations and future challenges of this area are also described.
- 332Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundbäck, T.; Nordlund, P.; Molina, D. M. The Cellular Thermal Shift Assay for Evaluating Drug Target Interactions in Cells. Nat. Protoc. 2014, 9 (9), 2100– 2122, DOI: 10.1038/nprot.2014.138[Crossref], [PubMed], [CAS], Google Scholar332https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht12msrfJ&md5=e7852e1a306bca1698a8e8463ae566b1The cellular thermal shift assay for evaluating drug target interactions in cellsJafari, Rozbeh; Almqvist, Helena; Axelsson, Hanna; Ignatushchenko, Marina; Lundbaeck, Thomas; Nordlund, Paer; Molina, Daniel MartinezNature Protocols (2014), 9 (9), 2100-2122CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Thermal shift assays are used to study thermal stabilization of proteins upon ligand binding. Such assays have been used extensively on purified proteins in the drug discovery industry and in academia to detect interactions. Recently, we published a proof-of-principle study describing the implementation of thermal shift assays in a cellular format, which we call the cellular thermal shift assay (CETSA). The method allows studies of target engagement of drug candidates in a cellular context, herein exemplified with exptl. data on the human kinases p38α and ERK1/2. The assay involves treatment of cells with a compd. of interest, heating to denature and ppt. proteins, cell lysis, and the sepn. of cell debris and aggregates from the sol. protein fraction. Whereas unbound proteins denature and ppt. at elevated temps., ligand-bound proteins remain in soln. We describe two procedures for detecting the stabilized protein in the sol. fraction of the samples. One approach involves sample workup and detection using quant. western blotting, whereas the second is performed directly in soln. and relies on the induced proximity of two target-directed antibodies upon binding to sol. protein. The latter protocol has been optimized to allow an increased throughput, as potential applications require large nos. of samples. Both approaches can be completed in a day.
- 333Franken, H.; Mathieson, T.; Childs, D.; Sweetman, G. M. A.; Werner, T.; Tögel, I.; Doce, C.; Gade, S.; Bantscheff, M.; Drewes, G.; Reinhard, F. B. M.; Huber, W.; Savitski, M. M. Thermal Proteome Profiling for Unbiased Identification of Direct and Indirect Drug Targets Using Multiplexed Quantitative Mass Spectrometry. Nat. Protoc. 2015, 10 (10), 1567– 1593, DOI: 10.1038/nprot.2015.101[Crossref], [PubMed], [CAS], Google Scholar333https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVyrtbzN&md5=d9c225616a35fc5dcc97db234452d7e1Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometryFranken, Holger; Mathieson, Toby; Childs, Dorothee; Sweetman, Gavain M. A.; Werner, Thilo; Toegel, Ina; Doce, Carola; Gade, Stephan; Bantscheff, Marcus; Drewes, Gerard; Reinhard, Friedrich B. M.; Huber, Wolfgang; Savitski, Mikhail M.Nature Protocols (2015), 10 (10), 1567-1593CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)The direct detection of drug-protein interactions in living cells is a major challenge in drug discovery research. Recently, the authors introduced an approach termed thermal proteome profiling (TPP), which enables the monitoring of changes in protein thermal stability across the proteome using quant. mass spectrometry. The authors detd. the intracellular thermal profiles for up to 7000 proteins, and by comparing profiles derived from cultured mammalian cells in the presence or absence of a drug it was possible to identify direct and indirect targets of drugs in living cells in an unbiased manner. Here the authors demonstrate the complete workflow using the histone deacetylase inhibitor panobinostat. The key to this approach is the use of isobaric tandem mass tag 10-plex (TMT10) reagents to label digested protein samples corresponding to each temp. point in the melting curve so that the samples can be analyzed by multiplexed quant. mass spectrometry. Important steps in the bioinformatic anal. include data normalization, melting curve fitting and statistical significance detn. of compd. concn.-dependent changes in protein stability. All anal. tools are made freely available as R and Python packages. The workflow can be completed in 2 wk.
- 334Komissarov, A. A.; Romanova, D. V.; Debabov, V. G. Complete Inactivation of Escherichia Coli Uridine Phosphorylase by Modification of Asp5 with Woodward’s Reagent K. J. Biol. Chem. 1995, 270 (17), 10050– 10055, DOI: 10.1074/jbc.270.17.10050[Crossref], [PubMed], [CAS], Google Scholar334https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXltlWns7w%253D&md5=5848107c614fd0e1adc180b05dc63e23Complete inactivation of Escherichia coli uridine phosphorylase by modification of Asp5 with Woodward's reagent KKomissarov, Andrey A.; Romanova, Darya V.; Debabov, Vladimir G.Journal of Biological Chemistry (1995), 270 (17), 10050-5CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site (Ki = 0.07 mM) with subsequent modification of a carboxyl (k2 = 1.2 min-1). Neither substrate alone protected uridine phosphorylase from inactivation. The presence of phosphate did not affect the Ki and k2 values. The addn. of uracil or uridine led to a significant increase of both Ki (to 2.5 or 2.1 mM, resp.) and k3 (to 6.1 or 4.8 min-1, resp.) values. Thus, WRK could react in accordance with slow (high affinity) and fast (low affinity) mechanisms. Combined addn. of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (Ser4-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys13). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp5 carboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Km values for phosphate and uridine were changed from 5.1 and 0.19 mM (or 7.3 and 0.14 mM according to Leer et al. (Leer, J. C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, resp. The hydroxamic enzyme had higher thermostability than the native enzyme. The results obtained demonstrated the importance of the carboxyl at position 5. The loss of activity after selective group replacement is due to impaired stabilization of the transition state rather than to a decline in substrate affinity or change of the active site structure.
- 335Qian, Y.; Schürmann, M.; Janning, P.; Hedberg, C.; Waldmann, H. Activity-Based Proteome Profiling Probes Based on Woodward’s Reagent K with Distinct Target Selectivity. Angew. Chem., Int. Ed. 2016, 55 (27), 7766– 7771, DOI: 10.1002/anie.201602666[Crossref], [CAS], Google Scholar335https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnsFWlt7k%253D&md5=2b544a0647a5c81b883e2be0791f04d9Activity-Based Proteome Profiling Probes Based on Woodward's Reagent K with Distinct Target SelectivityQian, Yong; Schuermann, Marc; Janning, Petra; Hedberg, Christian; Waldmann, HerbertAngewandte Chemie, International Edition (2016), 55 (27), 7766-7771CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Woodward's reagent K (WRK) is a reactive heterocyclic compd. that has been employed in protein chem. to covalently and unspecifically label proteins at nucleophilic amino acids, notably at histidine and cysteine. The authors have developed a panel of WRK-derived activity-based probes and show that surprisingly and unexpectedly, these probes are fairly selective for a few proteins in the human proteome. The WRK-derived probes show unique reactivity towards the catalytic N-terminal proline in the macrophage migration inhibitory factor (MIF) and can be used to label and, if equipped with a fluorophore, to image MIF activities in living cells.
- 336Harlow, K. W.; Switzer, R. L. Chemical Modification of Salmonella Typhimurium Phosphoribosylpyrophosphate Synthetase with 5′-(p-Fluorosulfonylbenzoyl)Adenosine. Identification of an Active Site Histidine. J. Biol. Chem. 1990, 265, 5487– 5493[PubMed], [CAS], Google Scholar336https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXitFejtLg%253D&md5=84e395a2c7b0d7ff965ba1659a791091Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidineHarlow, Kenneth W.; Switzer, Robert L.Journal of Biological Chemistry (1990), 265 (10), 5487-93CODEN: JBCHA3; ISSN:0021-9258.Liq. chromatog. procedures were developed for rapidly locating the site of reaction of chem. modification reagents with S. typhimurium 5-phosphoribosyl-α-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent, 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol FSBA/mol enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolyzates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence, Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was assocd. with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicated histidine-130 as the site of reaction with FSBA.
- 337Uchida, K.; Stadtman, E. R. Modification of Histidine Residues in Proteins by Reaction with 4-Hydroxynonenal. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (10), 4544– 4548, DOI: 10.1073/pnas.89.10.4544[Crossref], [PubMed], [CAS], Google Scholar337https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XksVeqtL4%253D&md5=e319c0a1200aa40784b00fde9bdd7cd6Modification of histidine residues in proteins by reaction with 4-hydroxynonenalUchida, Koji; Stadtman, E. R.Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (10), 4544-8CODEN: PNASA6; ISSN:0027-8424.Histidine residues in proteins are major targets for reaction with the lipid peroxidn. product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivs. upon subsequent treatment with NaB[3H]H4. Amino acid anal. of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stoichiometric formation of 3H-labeled amino acid hydrolyzates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the prodn. of two compds. Upon acid hydrolysis, both derivs. yielded stoichiometric amts. of histidine. However, after redn. with NaBH4, acid hydrolysis led to a mixt. of amino acid derivs. [presumably, isomeric forms of Nπ(Nτ)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addn. of the imidazole nitrogen atom of histidine to the α,β-unsatd. bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be detd.
- 338Yamaguchi, S.; Aldini, G.; Ito, S.; Morishita, N.; Shibata, T.; Vistoli, G.; Carini, M.; Uchida, K. Δ12-Prostaglandin J2 as a Product and Ligand of Human Serum Albumin: Formation of an Unusual Covalent Adduct at His146. J. Am. Chem. Soc. 2010, 132 (2), 824– 832, DOI: 10.1021/ja908878n[ACS Full Text], [CAS], Google Scholar338https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFKgu73J&md5=40501d528aa461e802e773930952b533Δ12-Prostaglandin J2 as a product and ligand of human serum albumin: Formation of an unusual covalent adduct at His146Yamaguchi, Satoru; Aldini, Giancarlo; Ito, Sohei; Morishita, Nozomi; Shibata, Takahiro; Vistoli, Giulio; Carini, Marina; Uchida, KojiJournal of the American Chemical Society (2010), 132 (2), 824-832CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Human serum albumin (HSA), the most abundant protein in plasma, has a very unique function, catalyzing the conversion of prostaglandin J2 (PGJ2), a dehydration product of PGD2, to yield Δ12-PGJ2. These PGD2 metabolites are actively transported into cells and accumulated in the nuclei, where they act as potent inducers of cell growth inhibition and cell differentiation, and exhibit their own unique spectrum of biol. effects. The facts that (i) arachidonic acid metabolites bind to human serum albumin (HSA) and the metab. of these mols. is altered as a result of binding, (ii) HSA catalyzes the transformation of PGJ2 into Δ12-PGJ2, and (iii) Δ12-PGJ2 is stable in serum suggest that HSA may bind and stabilize Δ12-PGJ2 in a specific manner. A mol. interaction anal. using surface plasmon resonance (Biacore) indeed suggested the presence of a specific Δ12-PGJ2-binding site in HSA. To investigate the mol. details of the binding of this PGD2 metabolite to albumin, we analyzed the cocrystal structure of the HSA-Δ12-PGJ2-myristate complex by X-ray crystallog. and found that two Δ12-PGJ2 mols. bind to a primary site in subdomain IB of the protein. The electron d. results suggested that one of the two Δ12-PGJ2 mols. that specifically bind to the site covalently interacted with a histidine residue (His146). Using nano-LC-MS/MS anal. of the HSA-Δ12-PGJ2 complex, the formation of an unusual Δ12-PGJ2-histidine adduct at His146 was confirmed. Thus, our crystallog. and mass spectrometric analyses of the HSA-Δ12-PGJ2 complex provided intriguing new insights into the mol. details of how this electrophilic ligand interacts with its primary producer and transporter.
- 339Yoshizawa, M.; Itoh, T.; Hori, T.; Kato, A.; Anami, Y.; Yoshimoto, N.; Yamamoto, K. Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands. J. Med. Chem. 2018, 61 (14), 6339– 6349, DOI: 10.1021/acs.jmedchem.8b00774[ACS Full Text], [CAS], Google Scholar339https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtF2htLzN&md5=5cc7c5b614f89aaa7582a0a25225e2e8Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic LigandsYoshizawa, Mami; Itoh, Toshimasa; Hori, Tatsuya; Kato, Akira; Anami, Yasuaki; Yoshimoto, Nobuko; Yamamoto, KeikoJournal of Medicinal Chemistry (2018), 61 (14), 6339-6349CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We designed and synthesized vitamin D analogs with an electrophile as covalent modifiers for the vitamin D receptor (VDR). Novel vitamin D analogs 1-4 have an electrophilic enone group at the side chain for conjugate addn. to His301 or His393 in the VDR. All compds. showed specific VDR-binding potency and agonistic activity. Covalent bond formations of 1-4 with the ligand-binding domain (LBD) of VDR were evaluated by electrospray ionization mass spectrometry. All compds. were shown to covalently bind to the VDR-LBD, and the abundance of VDR-LBD corresponding conjugate adducts of 1-4 increased with incubation time. Enone compds. 1 and 2 showed higher reactivity than the ene-ynone 3 and dienone 4 compds. Furthermore, we successfully obtained cocrystals of VDR-LBD with analogs 1-4. X-ray crystallog. anal. showed a covalent bond with His301 in VDR-LBD. We successfully synthesized vitamin D analogs that form a covalent bond with VDR-LBD.
- 340Liu, S.; Widom, J.; Kemp, C. W.; Crews, C. M.; Clardy, J. Structure of Human Methionine Aminopeptidase-2 Complexed with Fumagillin. Science 1998, 282 (5392), 1324– 1327, DOI: 10.1126/science.282.5392.1324[Crossref], [PubMed], [CAS], Google Scholar340https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXnsFenurY%253D&md5=729e37df06b4055f246753a544b6ee18Structure of human methionine aminopeptidase-2 complexed with fumagillinLiu, Shenping; Widom, Joanne; Kemp, Christopher W.; Crews, Craig M.; Clardy, JonScience (Washington, D. C.) (1998), 282 (5392), 1324-1327CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The fungal metabolite fumagillin suppresses the formation of new blood vessels, and a fumagillin analog is currently in clin. trials as an anticancer agent. The mol. target of fumagillin is methionine aminopeptidase-2 (MetAP-2). A 1.8 Å resoln. crystal structure of free and inhibited human MetAP-2 shows a covalent bond formed between a reactive epoxide of fumagillin and histidine-231 in the active site of MetAP-2. Extensive hydrophobic and water-mediated polar interactions with other parts of fumagillin provide addnl. affinity. Fumagillin-based drugs inhibit MetAP-2 but not MetAP-1, and the three-dimensional structure also indicates the likely determinants of this specificity. The structural basis for fumagillin's potency and specificity forms the starting point for structure-based drug design.
- 341Morgen, M.; Jöst, C.; Malz, M.; Janowski, R.; Niessing, D.; Klein, C. D.; Gunkel, N.; Miller, A. K. Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular Activity. ACS Chem. Biol. 2016, 11 (4), 1001– 1011, DOI: 10.1021/acschembio.5b00755[ACS Full Text], [CAS], Google Scholar341https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVGitbvO&md5=f83da1a8a3c4717b16cd0e90cc05f8a8Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular ActivityMorgen, Michael; Joest, Christian; Malz, Mona; Janowski, Robert; Niessing, Dierk; Klein, Christian D.; Gunkel, Nikolas; Miller, Aubry K.ACS Chemical Biology (2016), 11 (4), 1001-1011CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Methionine aminopeptidases (MetAPs) are responsible for the cotranslational cleavage of initiator methionines from nascent proteins. The MetAP2 subtype is up-regulated in many cancers, and selective inhibition of MetAP2 suppresses both vascularization and growth of tumors in animal models. The natural product fumagillin is a selective and potent irreversible inhibitor of MetAP2, and semisynthetic derivs. of fumagillin have shown promise in clin. studies for the treatment of cancer, and, more recently, for obesity. Further development of fumagillin derivs. has been complicated, however, by their generally poor pharmacokinetics. In an attempt to overcome these limitations, we developed an easily diversifiable synthesis of a novel class of MetAP2 inhibitors that were designed to mimic fumagillin's mol. scaffold but have improved pharmacol. profiles. These substances were found to be potent and selective inhibitors of MetAP2, as demonstrated in biochem. enzymic assays against three MetAP isoforms. Inhibitors with the same relative and abs. stereoconfiguration as fumagillin displayed significantly higher activity than their diastereomeric and enantiomeric isomers. X-ray crystallog. anal. revealed that the inhibitors covalently modify His231 in the MetAP2 active site via ring-opening of a spiroepoxide. Biochem. active substances inhibited the growth of endothelial cells and a MetAP2-sensitive cancer cell line, while closely related inactive isomers had little effect on the proliferation of either cell type. These effects correlated with altered N-terminal processing of the protein 14-3-3-γ. Finally, selected substances were found to have improved stabilities in mouse plasma and microsomes relative to the clin. investigated fumagillin deriv. beloranib.
- 342Jakob, C. G.; Upadhyay, A. K.; Donner, P. L.; Nicholl, E.; Addo, S. N.; Qiu, W.; Ling, C.; Gopalakrishnan, S. M.; Torrent, M.; Cepa, S. P.; Shanley, J.; Shoemaker, A. R.; Sun, C. C.; Vasudevan, A.; Woller, K. R.; Shotwell, J. B.; Shaw, B.; Bian, Z.; Hutti, J. E. Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315. J. Med. Chem. 2018, 61 (15), 6647– 6657, DOI: 10.1021/acs.jmedchem.8b00305[ACS Full Text], [CAS], Google Scholar342https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlWrtrbF&md5=15c54988cd3080d5a675badbb62b6fecNovel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315Jakob, Clarissa G.; Upadhyay, Anup K.; Donner, Pamela L.; Nicholl, Emily; Addo, Sadiya N.; Qiu, Wei; Ling, Christopher; Gopalakrishnan, Sujatha M.; Torrent, Maricel; Cepa, Steven P.; Shanley, Jason; Shoemaker, Alexander R.; Sun, Chaohong C.; Vasudevan, Anil; Woller, Kevin R.; Shotwell, J. Brad; Shaw, Bailin; Bian, Zhiguo; Hutti, Jessica E.Journal of Medicinal Chemistry (2018), 61 (15), 6647-6657CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)IDH1 plays a crit. role in a no. of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochem. characterization of two novel inhibitors of wild-type IDH1. In addn., we present the first ligand-bound crystallog. characterization of these novel small mol. IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsatd. enone 1 makes a unique covalent linkage through active site H315. As few small mols. have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small mol. design.
- 343Lin, S.; Yang, X.; Jia, S.; Weeks, A. M.; Hornsby, M.; Lee, P. S.; Nichiporuk, R. V.; Iavarone, A. T.; Wells, J. A.; Toste, F. D.; Chang, C. J. Redox-Based Reagents for Chemoselective Methionine Bioconjugation. Science 2017, 355 (6325), 597– 602, DOI: 10.1126/science.aal3316[Crossref], [PubMed], [CAS], Google Scholar343https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXit1Okur4%253D&md5=90957bb9f95794e12417ade39bebd734Redox-based reagents for chemoselective methionine bioconjugationLin, Shixian; Yang, Xiaoyu; Jia, Shang; Weeks, Amy M.; Hornsby, Michael; Lee, Peter S.; Nichiporuk, Rita V.; Iavarone, Anthony T.; Wells, James A.; Toste, F. Dean; Chang, Christopher J.Science (Washington, DC, United States) (2017), 355 (6325), 597-602CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In contrast, selective ligation to the other sulfur-contg. amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, the authors report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. The authors highlight the broad utility of this conjugation method to enable precise addn. of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes.
- 344Bizet, V.; Hendriks, C. M. M.; Bolm, C. Sulfur Imidations: Access to Sulfimides and Sulfoximines. Chem. Soc. Rev. 2015, 44 (11), 3378– 3390, DOI: 10.1039/C5CS00208G[Crossref], [PubMed], [CAS], Google Scholar344https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXns1yisrw%253D&md5=f783729be2df3166e6e3b09c3b6d311fSulfur imidations: access to sulfimides and sulfoximinesBizet, Vincent; Hendriks, Christine M. M.; Bolm, CarstenChemical Society Reviews (2015), 44 (11), 3378-3390CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Being mono-aza analogs of sulfoxides and sulfones, sulfimides and sulfoximines, resp., are important compds. in asym. synthesis, crop protection and medicinal chem. various methods were developed for their prepn. For their prepn. various methods were developed. In the search for the optimal synthetic approach for a given target compd., several parameters have to be considered which also include safety issues and availability of starting materials. An overview of sulfur imidation methods, classified by imidating agents and compds. with a related behavior were reviewed. The aim of this survey was to provide a practical ''tool box'' for the synthetic chemist by mapping the advantages and disadvantages assocd. with the use of these compds.
- 345Gong, Y.; Andina, D.; Nahar, S.; Leroux, J.-C.; Gauthier, M. A. Releasable and Traceless PEGylation of Arginine-Rich Antimicrobial Peptides. Chem. Sci. 2017, 8 (5), 4082– 4086, DOI: 10.1039/C7SC00770A[Crossref], [PubMed], [CAS], Google Scholar345https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsVakur4%253D&md5=ce26e313c6d8779e5c48a86d80eb47efReleasable and traceless PEGylation of arginine-rich antimicrobial peptidesGong, Y.; Andina, D.; Nahar, S.; Leroux, J.-C.; Gauthier, M. A.Chemical Science (2017), 8 (5), 4082-4086CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Arginine-rich antimicrobial peptides (AMPs) are emerging therapeutics of interest. However, their applicability is limited by their short circulation half-life, caused in part by their small size and digestion by blood proteases. This study reports a strategy to temporarily mask arginine residues within AMPs with methoxy poly(ethylene glycol). Based on the reagent used, release of AMPs occurred in hours to days in a completely traceless fashion. In vitro, conjugates were insensitive to serum proteases, and released native AMP with full in vitro bioactivity. This strategy is thus highly relevant and should be adaptable to the entire family of arginine-rich AMPs. It may potentially be used to improve AMP-therapies by providing a more steady concn. of AMP in the blood after a single injection, avoiding toxic effects at high AMP doses, and reducing the no. of doses required over the treatment duration.
- 346Seki, Y.; Ishiyama, T.; Sasaki, D.; Abe, J.; Sohma, Y.; Oisaki, K.; Kanai, M. Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins. J. Am. Chem. Soc. 2016, 138 (34), 10798– 10801, DOI: 10.1021/jacs.6b06692[ACS Full Text], [CAS], Google Scholar346https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlOgt7%252FO&md5=1dd578dc20df2d1092694c474a4c2c4dTransition Metal-Free Tryptophan-Selective Bioconjugation of ProteinsSeki, Yohei; Ishiyama, Takashi; Sasaki, Daisuke; Abe, Junpei; Sohma, Youhei; Oisaki, Kounosuke; Kanai, MotomuJournal of the American Chemical Society (2016), 138 (34), 10798-10801CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Chem. modifications of native proteins can facilitate prodn. of supernatural protein functions that are not easily accessible by complementary methods relying on genetic manipulations. However, accomplishing precise control over selectivity while maintaining structural integrity and homogeneity still represents a formidable challenge. Herein, the authors report a transition metal-free method for tryptophan-selective bioconjugation of proteins that is based on an organoradical and operates under ambient conditions. This method exhibits low levels of cross-reactivity and leaves higher-order structures of the protein and various functional groups therein unaffected. The strategy to target less abundant amino acids contributes to the formation of structurally homogeneous conjugates, which may even be suitable for protein crystallog. The absence of toxic metals and biochem. incompatible conditions allows a rapid functional modulation of native proteins such as antibodies and pathogenic aggregative proteins, and this method may thus easily find therapeutic applications.
- 347Shibata, Y.; Chiba, M. The Role of Extrahepatic Metabolism in the Pharmacokinetics of the Targeted Covalent Inhibitors Afatinib, Ibrutinib, and Neratinib. Drug Metab. Dispos. 2015, 43 (3), 375– 384, DOI: 10.1124/dmd.114.061424[Crossref], [PubMed], [CAS], Google Scholar347https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXit1OmsrY%253D&md5=eaac67ce7cea5f2432c5144922ba3dd2The role of extrahepatic metabolism in the pharmacokinetics of the targeted covalent inhibitors afatinib, ibrutinib, and neratinibShibata, Yoshihiro; Chiba, MasatoDrug Metabolism & Disposition (2015), 43 (3), 375-384, 10 pp.CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)Despite the fact that much progress has been made recently in the development of targeted covalent inhibitors (TCIs), their pharmacokinetics (PK) have not been well characterized in the light of extrahepatic clearance (CLextH) by glutathione (GSH)/glutathione S-transferase (GST)-dependent conjugation attributable to the unique electrophilic structure (e.g., acrylamide moiety) of TCI compds. In the present study, CLextH values were examd. in rat, dog, and monkey to predict the contribution of CLextH to the PK of the TCIs afatinib, ibrutinib, and neratinib in humans. Afatinib and neratinib both underwent extensive conjugation with GSH in buffer and cytosol fractions of liver and kidney, whereas ibrutinib showed much lower reactivity/susceptibility to GSH/GST-dependent conjugation. The CLextH in each species was calcd. from the difference between obsd. total body clearance and predicted hepatic clearance (CLH) in cryopreserved hepatocytes suspended in 100% serum of the corresponding species. The power-based simple allometry relating the CLextH for the unbound compd. to animal body wt. was applicable across species for afatinib and neratinib (R2 ≥ 0.9) but not for ibrutinib (R2 = 0.04). The predicted AUC after oral administration of afatinib and neratinib agreed reasonably closely with reported values in phase I dose-escalation studies. Comparisons of CLextH and CLH predicted that CLextH largely detd. the PK of afatinib (>90% as a proportion of total body clearance) and neratinib (∼34%) in humans. The present method can serve as one of the tools for the optimization of PK in humans at the discovery stage for the development of TCI candidates.
- 348Scheers, E.; Leclercq, L.; de Jong, J.; Bode, N.; Bockx, M.; Laenen, A.; Cuyckens, F.; Skee, D.; Murphy, J.; Sukbuntherng, J.; Mannens, G. Absorption, Metabolism, and Excretion of Oral 14C Radiolabeled Ibrutinib: An Open-Label, Phase I, Single-Dose Study in Healthy Men. Drug Metab. Dispos. 2015, 43, 289– 297, DOI: 10.1124/dmd.114.060061[Crossref], [PubMed], [CAS], Google Scholar348https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpt1ansg%253D%253D&md5=1f1e728078220c9d7cd973d4992c7308Absorption, metabolism, and excretion of pral 14C radiolabeled ibrutinib: an open-label, phase I, single-dose study in healthy menScheers, Ellen; Leclercq, Laurent; de Jong, Jan; Bode, Nini; Bockx, Marc; Laenen, Aline; Cuyckens, Filip; Skee, Donna; Murphy, Joe; Sukbuntherng, Juthamas; Mannens, GeertDrug Metabolism & Disposition (2015), 43 (2), 289-297, 9 pp.CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)The absorption, metab., and excretion of ibrutinib were investigated in healthy men after administration of a single oral dose of 140 mg of 14C-labeled ibrutinib. The mean S.D. cumulative excretion of radioactivity of the dose was 7.8% 1.4% in urine and 80.6% 3.1% in feces with <1% excreted as parent ibrutinib. Only oxidative metabolites and very limited parent compd. were detected in feces, and this indicated that ibrutinib was completely absorbed from the gastrointestinal tract. Metab. occurred via three major pathways hydroxylation of the Ph M35, opening of the piperidine M25 and M34, and epoxidn. of the ethylene on the acryloyl moiety with further hydrolysis to dihydrodiol PCI-45227, and M37. Addnl. metabolites were formed by combinations of the primary metabolic pathways or by further metab. In blood and plasma, a rapid initial decline in radioactivity was obsd. along with long terminal elimination half-life for total radioactivity. The max. concn. Cmax and area under the concn.-time curve AUC for total radioactivity were higher in plasma compared with blood. The main circulating entities in blood and plasma were M21 sulfate conjugate of a monooxidized metabolite on phenoxyphenyl, M25, M34, M37 PCI-45227, and ibrutinib. At Cmax of radioactivity, 12% of total radioactivity was accounted for by covalent binding in human plasma. More than 50% of total plasma radioactivity was attributed to covalently bound material from 8 h onward; as a result, covalent binding accounted for 38% and 51% of total radioactivity AUC0-24 h and AUC0-72 h, resp. No effect of CYP2D6 genotype was obsd. on ibrutinib metab. Ibrutinib was well-tolerated by healthy participants.
- 349Chatterjee, P.; Botello-Smith, W. M.; Zhang, H.; Qian, L.; Alsamarah, A.; Kent, D.; Lacroix, J. J.; Baudry, M.; Luo, Y. Can Relative Binding Free Energy Predict Selectivity of Reversible Covalent Inhibitors?. J. Am. Chem. Soc. 2017, 139 (49), 17945– 17952, DOI: 10.1021/jacs.7b08938[ACS Full Text], [CAS], Google Scholar349https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslyrs7bI&md5=e6a38ff570134e956eb43bd4a874ca55Can Relative Binding Free Energy Predict Selectivity of Reversible Covalent Inhibitors?Chatterjee, Payal; Botello-Smith, Wesley M.; Zhang, Han; Qian, Li; Alsamarah, Abdelaziz; Kent, David; Lacroix, Jerome J.; Baudry, Michel; Luo, YunJournal of the American Chemical Society (2017), 139 (49), 17945-17952CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Reversible covalent inhibitors have many clin. advantages over noncovalent or irreversible covalent drugs. However, apart from selecting a warhead, substantial efforts in design and synthesis are needed to optimize noncovalent interactions to improve target-selective binding. Computational prediction of binding affinity for reversible covalent inhibitors presents a unique challenge since the binding process consists of multiple steps, which are not necessarily independent of each other. In this study, the authors lay out the relation between relative binding free energy and the overall reversible covalent binding affinity using a two-state binding model. To prove the concept, the authors employed free energy perturbation (FEP) coupled with λ-exchange mol. dynamics method to calc. the binding free energy of a series of α-ketoamide analogs relative to a common warhead scaffold, in both noncovalent and covalent binding states, and for two highly homologous proteases, calpain-1 and calpain-2. The authors conclude that covalent binding state alone, in general, can be used to predict reversible covalent binding selectivity. However, exceptions may exist. Therefore, the authors also discuss the conditions under which the noncovalent binding step is no longer negligible and propose to combine the relative FEP calcns. with a single QM/MM calcn. of warhead to predict the binding affinity and binding kinetics. The FEP calcns. also revealed that covalent and noncovalent binding states of an inhibitor do not necessarily exhibit the same selectivity. Thus, investigating both binding states, as well as the kinetics will provide extremely useful information for optimizing reversible covalent inhibitors.
- 350Alberty, R. A.; Hammes, G. G. Application of the Theory of Diffusion-Controlled Reactions to Enzyme Kinetics. J. Phys. Chem. 1958, 62 (2), 154– 159, DOI: 10.1021/j150560a005[ACS Full Text], [CAS], Google Scholar350https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG1cXlsVSqtg%253D%253D&md5=140ac5d97b80b78b09c1a8523a7c1df7Application of the theory of diffusion-controlled reactions to enzyme kineticsAlberty, Robert A.; Hammes, Gordon G.Journal of Physical Chemistry (1958), 62 (), 154-9CODEN: JPCHAX; ISSN:0022-3654.cf. C.A. 51, 9757h. Extrapolation of the min. values of the 2nd-order rate consts. for the reaction of fumarate ions with fumarase to zero ionic strength yields a value of about 3 × 1010/sec. mole at 25°. The magnitude of this rate const. can be accounted for theoretically by considering the diffusion of substrate into a hemispherical reaction site on a plane with a reaction radius of 5 A., provided that the effect of elec. charges in increasing the rate is included. It is presumed that the bimol. reaction of fumarase with its substrates is diffusion controlled. The effect of electrolyte concn. on the experimentally detd. min. values of the 2nd-order rate consts. indicates a charge of +2 to +3 on the enzymic site. The application of these theoretical considerations to enzymic reactions, in general, is discussed for the case where the effect of protein charges outside the enzymic site may be neglected.
- 351Wright, M. H.; Sieber, S. A. Chemical Proteomics Approaches for Identifying the Cellular Targets of Natural Products. Nat. Prod. Rep. 2016, 33 (5), 681– 708, DOI: 10.1039/C6NP00001K[Crossref], [PubMed], [CAS], Google Scholar351https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsF2gsbc%253D&md5=0732834925092e013d9545f46ce4fd2fChemical proteomics approaches for identifying the cellular targets of natural productsWright, M. H.; Sieber, S. A.Natural Product Reports (2016), 33 (5), 681-708CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)Covering: 2010 up to 2016: Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chem. and biol. today. Chem. proteomics is a growing area of chem. biol. that seeks to design small mol. probes to understand protein function. In the context of natural products, chem. proteomics can be used to identify the protein binding partners or targets of small mols. in live cells. Here, the authors highlight recent examples of chem. probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chem. reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. The authors also focus here on strategies that employ a click reaction, the copper-catalyzed azide-alkyne cycloaddn. reaction (CuAAC), to allow minimal functionalization of natural product scaffolds with an alkyne or azide tag. The authors also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterized chem. probe for binding to a particular set of protein targets. Fueled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quant. proteomics to help define the true interacting partners of probes, and the authors highlight the opportunities this rapidly evolving technol. provides in chem. proteomics. Finally, some of the limitations and challenges of chem. proteomics approaches are discussed.